Aflatoxins - Biochemistry and Molecular Biology 978-953-307-395-8

Citation preview

AFLATOXINS – BIOCHEMISTRY AND MOLECULAR BIOLOGY Edited by Ramón Gerardo Guevara-González

Aflatoxins – Biochemistry and Molecular Biology Edited by Ramón Gerardo Guevara-González

Published by InTech Janeza Trdine 9, 51000 Rijeka, Croatia Copyright © 2011 InTech All chapters are Open Access articles distributed under the Creative Commons Non Commercial Share Alike Attribution 3.0 license, which permits to copy, distribute, transmit, and adapt the work in any medium, so long as the original work is properly cited. After this work has been published by InTech, authors have the right to republish it, in whole or part, in any publication of which they are the author, and to make other personal use of the work. Any republication, referencing or personal use of the work must explicitly identify the original source. Statements and opinions expressed in the chapters are these of the individual contributors and not necessarily those of the editors or publisher. No responsibility is accepted for the accuracy of information contained in the published articles. The publisher assumes no responsibility for any damage or injury to persons or property arising out of the use of any materials, instructions, methods or ideas contained in the book. Publishing Process Manager Petra Nenadic Technical Editor Teodora Smiljanic Cover Designer Jan Hyrat Image Copyright Juan Gaertner, 2010. Used under license from Shutterstock.com First published September, 2011 Printed in Croatia A free online edition of this book is available at www.intechopen.com Additional hard copies can be obtained from [email protected]

Aflatoxins – Biochemistry and Molecular Biology, Edited by Ramón Gerardo Guevara-González p. cm. ISBN 978-953-307-395-8

free online editions of InTech Books and Journals can be found at www.intechopen.com

Contents Preface IX Part 1

Biosynthesis and Biotransformation

1

Chapter 1

Biotransformation of Aflatoxin B1 and Its Relationship with the Differential Toxicological Response to Aflatoxin in Commercial Poultry Species 3 Gonzalo J. Diaz and Hansen W. Murcia

Chapter 2

Control of Aflatoxin Biosynthesis in Aspergilli 21 Kenneth C. Ehrlich, Perng-Kuang Chang, Jiujiang Yu, Jeffrey W. Cary and Deepak Bhatnagar

Chapter 3

Aflatoxin Biosynthetic Pathway and Pathway Genes Jiujiang Yu and Kenneth C. Ehrlich

Chapter 4

Conserved Regulatory Mechanisms Controlling Aflatoxin and Sterigmatocystin Biosynthesis 67 Ana M. Calvo and Sourabh Dhingra

Part 2

Biomarkers and Breeding

41

89

Chapter 5

Identification of Gene Markers in Aflatoxin-Resistant Maize Germplasm for Marker-Assisted Breeding 91 Robert L. Brown, Abebe Menkir, Zhi-Yuan Chen, Meng Luo and Deepak Bhatnagar

Chapter 6

Biomarkers of Aflatoxin Exposure and Its Relationship with the Hepatocellular Carcinoma 107 Alessandra Vincenzi Jager, Fernando Silva Ramalho, Leandra Náira Zambelli and Carlos Augusto Fernandes Oliveira

Chapter 7

The Use of Proteomics as a Novel Tool in Aflatoxin Research 127 E. Razzazi-Fazeli, M. Rizwan, C. Mayrhofer and K. Nöbauer

VI

Contents

Chapter 8

Part 3 Chapter 9

Genetic Resistance to Drought in Maize and Its Relationship in Aflatoxins Production 151 Ramón Gerardo Guevara-González, Angela María Chapa-Oliver, Laura Mejía-Teniente, Irineo Torres-Pacheco, Moises Alejandro Vazquez-Cruz, Juan Jesús Cervantes-Landaverde, Ricardo Ernesto Preciado-Ortiz and Ernesto Moreno- Martinez Pathogenesis and Toxicology 161 The Molecular Pathogenesis of Aflatoxin with Hepatitis B Virus-Infection in Hepatocellular Carcinoma 163 Hai-Xia Cao and Jian-Gao Fan

Chapter 10

A Comprehensive Review of Male Reproductive Toxic Effects of Aflatoxin 177 Mohammad A. Akbarsha, Faisal Kunnathodi and Ali A. Alshatwi

Chapter 11

Aflatoxicosis in Layer and Breeder Hens Milad Manafi

Chapter 12

Aflatoxins and Aflatoxicosis in Human and Animals D. Dhanasekaran, S. Shanmugapriya, N. Thajuddin and A. Panneerselvam

Chapter 13

Aflatoxins and Their Impact on Human and Animal Health: An Emerging Problem 255 Eva G. Lizárraga-Paulín, Ernesto Moreno-Martínez and Susana P. Miranda-Castro

Part 4

203

221

Miscellaneous 283

Chapter 14

Aflatoxins: Mechanisms of Inhibition by Antagonistic Plants and Microorganisms 285 Mehdi Razzaghi-Abyaneh, Masoomeh Shams-Ghahfarokhi and Perng-Kuang Chang

Chapter 15

The Evolutionary Dynamics in the Research on Aflatoxins During the 2001-2010 Decade 305 Martín G. Theumer and Héctor R. Rubinstein

Chapter 16

Binding of Aflatoxin B1 to Lactic Acid Bacteria and Saccharomyces cerevisiae in vitro: A Useful Model to Determine the Most Efficient Microorganism 323 Romina P. Pizzolitto, Dante J. Bueno, María R. Armando, Lilia Cavaglieri, Ana M. Dalcero and Mario A. Salvano

Chapter 17

The Population Dynamics of Aflatoxigenic Aspergilli Geromy G. Moore, Shannon B. Beltz, Ignazio Carbone, Kenneth C. Ehrlich and Bruce W. Horn

347

Contents

Chapter 18

Aflatoxin in Agricultural Commodities and Herbal Medicine 367 Mehrdad Tajkarimi, Mohammad Hossein Shojaee, Hassan Yazdanpanah and Salam A. Ibrahim

Chapter 19

A Review of Aflatoxin M1, Milk, and Milk Products 397 Hamid Mohammadi

Chapter 20

Aflatoxins: Contamination, Analysis and Control 415 Giniani Carla Dors, Sergiane Souza Caldas, Vivian Feddern, Renata Heidtmann Bemvenuti, Helen Cristina dos Santos Hackbart, Michele Moraes de Souza, Melissa dos Santos Oliveira, Jaqueline Garda-Buffon, Ednei Gilberto Primel and Eliana Badiale-Furlong

Chapter 21

Estimated Daily Intake of Aflatoxin M1 in Thailand 439 Nongluck Ruangwises, Piyawat Saipan and Suthep Ruangwises

Chapter 22

Influence of Soluble Feed Proteins and Clay Additive Charge Density on Aflatoxin Binding in Ingested Feeds William F. Jaynes and Richard E. Zartman

447

VII

Preface Aflatoxins are produced by Aspergillus flavi group species and are thought to be one of the most cancerous natural substances known. Economically and biologically the most important fungal species able to produce the aflatoxins are Aspergillus flavus and Aspergillus parasiticus. The biosynthesis of aflatoxins, as all secondary metabolites, is strongly dependent on growth conditions such as substrate composition or physical factors like pH, water activity, temperature or modified atmospheres. Depending on the particular combination of external growth parameters the biosynthesis of aflatoxin can either be completely inhibited, albeit normal growth is still possible or the biosynthesis pathway can be fully activated. Knowledge about these relationships enables an assessment of which parameter combinations can control aflatoxin biosynthesis or which are conducive to phenotypic aflatoxin production. All these aspects are fascinating and strongly growing in knowledge based on the work of excellent researchers as those invited to write every chapter presented in this book. Finally, this book is an attempt to provide a wide and current approach of top studies in aflatoxins biochemistry and molecular biology, as well as some general aspects to researchers interested in this field.

Dr. Ramon Gerardo Guevara-Gonzalez Professor Biosystems Engineering Group School of Engineering Queretaro Autonomous University Queretaro, Qro, Mexico

Part 1 Biosynthesis and Biotransformation

1 Biotransformation of Aflatoxin B1 and Its Relationship with the Differential Toxicological Response to Aflatoxin in Commercial Poultry Species Gonzalo J. Diaz and Hansen W. Murcia Facultad de Medicina Veterinaria y de Zootecnia Universidad Nacional de Colombia, Bogotá D.C., Colombia 1. Introduction Aflatoxin B1 (AFB1) is a highly toxic compound (LD50 = 1-50 mg/kg) for most animal species, although it is extremely toxic (LD50 < 1 mg/kg) for some highly susceptible species such as pigs, dogs, cats, rainbow trouts, and ducklings. The toxic effects of AFB1 are both dose and time dependent and two distinct forms of aflatoxicosis, namely acute and chronic, can be distinguished depending on the level and length of time of aflatoxin exposure. In many species acute poisoning is characterized by an acute hepatotoxic disease that manifests itself with depression, anorexia, icterus, and hemorrhages. Histologic hepatic lesions include periportal necrosis associated with bile duct proliferation and oval cell hyperplasia. Chronic aflatoxicosis resulting from regular low-level dietary intake of aflatoxins causes unspecific signs such as reduced weight gain, reduced feed intake, and reduced feed conversion in pigs and poultry, and reduced milk yield in cows. Another effect of chronic exposure is aflatoxin-induced hepatocellular carcinoma, bile duct hyperplasia and hepatic steatosis (fatty liver). However, these effects are species-specific and not all animals exposed to aflatoxin develop liver cancer. For example, the only poultry species that develops hepatocellular carcinoma after AFB1 exposure is the duck. Differences in the susceptibility to acute and chronic AFB1 toxicosis have been observed among animals of different species. Animals having the highest sensitivity are the duckling, piglet, rabbit, dog and cat, while chickens, mice, hamsters, and chinchillas are relatively resistant. Further, mature animals are generally more resistant to AFB1 than young ones and females are more resistant than males. In general, in commercial poultry species, intake of feed contaminated with AFB1 results primarily in liver damage (the target organ of AFB1 is the liver), associated with immunosuppression, poor performance, and even mortality when the dietary levels are high enough. However, there is wide variability in specific species sensitivity to AFB1 and the susceptibility ranges from ducklings > turkey poults > goslings > pheasant chicks > quail chicks > chicks (Leeson et al., 1995). Even though there is still no clear explanation for this differential sensitivity, differences in susceptibility could be due to differences in AFB1 biotransformation pathways among species. The aim of the present chapter is to review the current knowledge on AFB1 biotransformation, with emphasis on

4

Aflatoxins – Biochemistry and Molecular Biology

commercial poultry species, and to correlate this information with the in vivo susceptibility to AFB1 in these species.

2. Biotransformation of aflatoxin B1 In general, the metabolism or biotransformation of xenobiotics (chemicals foreign to the organism) is a process aimed at converting the original molecules into more hydrophilic compounds readily excretable in the urine (by the kidney) or in the bile (by the liver). It has traditionally been conceptualized that this process occurs in two phases known as Phase I and Phase II, although some authors argue that this classification is no longer tenable and should be eliminated (Josephy et al., 2005). Phase I metabolism consists mainly of enzyme-mediated hydrolysis, reduction and oxidation reactions, while Phase II metabolism involves conjugation reactions of the original compound or the compound modified by a previous Phase I reaction. The current state of knowledge on the metabolism of AFB1 in different avian and mammalian species is summarized in Figure 1. As Figure 1 shows, a wide array of metabolites can be O

O O 2

8

O

9a

6a

4

O Cytosolic NADPH2 reductase

H

O

O CH3

5

Aflatoxin Q1

O CH3

Aflatoxicol H1 O

O

H HO

O

CYP450s

O O

O

O

O

OH O

O CH3

Aflatoxin B1 Cytosolic/red blood cell dehydrogenase

OH

H

O

OH O

OH

O

Cytosolic NADPH2 reductase

3

O

O

O

O

O

9

O CH3

O CH3 O

Aflatoxin B2a

O

Aflatoxicol O

O O O

O

O

O

OH

OH

H2 N

O O

O-Gluc

OH

O

UGT

O

O

OH

O O

O

O

O O

OH

O

HO

O-Gluc

Aflatoxin M1-P1-glucuronide

Aflatoxin M1-P1

N COOH

OH

Aflatoxin P1-glucuronide

O

OH S

O

O

O

O

O

O CH3

CYP450

O

Spontaneous hydrolysis or EPHX

OH

Aflatoxicol M1

O

O

O

O

O CH3

Aflatoxin M1 O O

GST-M1 (primates) GST-A1 (mouse)

OH

O

Microsomal O dehydrogenase

O

UGT

O CH3

Aflatoxin B1 exo8,9-epoxide COOH

Cytosolic NADPH2 reductase

O

O

Aflatoxin P1

O O

O

O

O

O O

O CH3

O

O

O CH3

O

O

Aflatoxin B1 exo8,9-dihidrodiol

O

O

O

HO

O

AFAR O

O

OH

OH

Aflatoxin B1 exoglutathion

O CH3

Dialdehyde phenolate

HO O

O CH3

Dialcohol phenolate

Fig. 1. Biotransformation reactions of aflatoxin B1 in poultry and mammals, including humans. The main CYP450s involved in these reactions are CYP1A1, CYP1A2, CYP2A6 and CYP3A4. Not all reactions occur in a single species.

Biotransformation of Aflatoxin B1 and Its Relationship with the Differential Toxicological Response to Aflatoxin in Commercial Poultry Species

5

produced directly from AFB1 (by oxidation and reduction reactions) or indirectly by further biotransformation of the metabolites formed. However, not all of these reactions occur in a single species and, in fact, only a few of them have been reported in poultry. Most AFB1 Phase I reactions are oxidations catalyzed by cytochrome P450 (CYP450) enzymes, but one reaction is catalyzed by a cytosolic reductase, corresponding to the reduction of AFB1 to aflatoxicol (AFL). Phase II reactions are limited to conjugation of the metabolite AFB1-exo-8,9-epoxide (AFBO) with glutathione (GSH, -glutamyl-cysteinyl-glycine), and conjugation of aflatoxins P1 and M1-P1 with glucuronic acid. Conjugation of AFBO with GSH is a nucleophilic trapping process catalyzed by specific glutathione transferase (GST) enzymes. The AFBO may also be hydrolyzed by an epoxide hydrolase (EPHX) to form AFB1-exo-8,9-dihydrodiol, although this reaction may also occur spontaneously. The dihydrodiol is in equilibrium with the dialdehyde phenolate form, which can be reduced by AFB1 aldehyde reductase (AFAR), an enzyme that catalyzes the NADPH-dependent reduction of the dialdehyde to dialcohol phenolate (Guengerich et al., 2001). The translocation of xenobiotics across cell membranes by specific proteins known as transporters has been termed by some as “Phase III” metabolism. However, this process does not involve any modification of the xenobiotic structure and therefore it cannot be termed metabolism. This process, however, may have important implications on the toxic effect of a xenobiotic, particularly if the specific transporter involved in the translocation of the compound is not expressed normally, presents a genetic abnormality or becomes saturated. One transporter that has been identified as responsible for the translocation of a mycotoxin from the sinusoidal hepatic space into the hepatocyte is OATP (organic anion transporter polypeptide), which transports ochratoxin A (Diaz, 2000). However, no transporters for AFB1 have yet been described. 2.1 Phase I metabolism of aflatoxin B1 As mentioned before, the Phase I metabolism of AFB1 is carried out mainly by members of the CYP450 superfamily of enzymes. Their name comes from the absorption maximum at 450 nm when the reduced form complexes with carbon monoxide (Omura & Sato, 1964). CYP450s are membrane bound enzymes that can be isolated in the so-called microsomal fraction which is formed from endoplasmic reticulum when the cell is homogenized and fractionated by differential ultracentrifugation; microsomal vesicles are mainly fragments of the endoplasmic reticulum in which most of the enzyme activity is retained. The highest concentration of CYP450s involved in xenobiotic biotransformation is found in the endoplasmic reticulum of hepatocytes but CYP450s are present in virtually every tissue. CYP450s are classified into families identified by a number (e.g., 1, 2, 3, and 4), subfamilies identified by a letter (e.g., 2A, 2B, 2D, and 2E), and individual members identified by another number (e.g. CYP2A6, CYP2E1). Collectively, CYP450 enzymes participate in a variety of oxidative reactions with lipophilic xenobiotics and endogenous substrates including hydroxylation of an aliphatic or aromatic carbon, epoxidation of a double bond, heteroatom (S-, N- and I-) oxygenation and Nhydroxylation, heteroatom (O-, S-, and N-) dealkylation, oxidative group transfer, cleavage of esters, and dehydrogenation (Parkinson & Ogilvie, 2008). In regards to AFB1, CYP450s can hydroxylate, hydrate, O-demethylate, and epoxidate the molecule. 2.1.1 Hydroxylation and hydration of aflatoxin B1 CYP450s can produce at least three monohydroxylated metabolites from AFB1, namely aflatoxins M1 (AFM1), Q1 (AFQ1), and B2a (AFB2a) (Fig. 1). AFM1 was first isolated from the

6

Aflatoxins – Biochemistry and Molecular Biology

milk of cows and rats fed AFB1-contaminated peanut meal and it was initially termed “milk toxin” (de Iongh et al., 1964). It was later discovered that AFM1 is not a metabolite exclusive of mammals and, in fact, it is produced by crude or isolated microsomal liver preparations from many non-mammalian species. For example, AFM1 was found in most tissues of chickens receiving a diet containing 2,057 ppb AFB1 for 35 days (Chen et al., 1984); the highest level was found in the liver and kidneys, which relates to the important role of these organs in the biotransformation and elimination of xenobiotics, respectively. AFQ1 results from the 3α-hydroxylation of AFB1 and it was first discovered as a major metabolite of AFB1 from monkey liver microsomal incubations (Masri et al., 1974). The predominant enzyme responsible for AFQ1 formation in human liver microsomes is CYP3A4 (Raney et al., 1992b) and AFQ1 is considered to be a major metabolite of AFB1 in humans and monkeys in vitro (Hsieh et al., 1974). Although AFQ1 has been detected as a minor metabolite of chicken and duck microsomal preparations (Leeson et al., 1995) it is considered to be a significant detoxication pathway of AFB1 (Raney et al., 1992b). In fact, AFQ1 is about 18 times less toxic for chicken embryos than AFB1 and it is not mutagenic in the Salmonella typhimurium TA 1538 test (Hsieh et al., 1974). The hydration of the vinyl ether double bond (C8-C9) of AFB1 produces the 8-hydroxy derivative or hemiacetal, also known as AFB2a. This metabolite was discovered in 1966 and, interestingly, it can be produced enzymatically (by both higher organisms and microbial metabolism), by photochemical degradation of AFB1, and by the treatment of AFB1 with acid (Lillehoj & Ciegler, 1969). The formation of the hemiacetal is difficult to assess in vitro because of strong protein binding, which probably involves the formation of Schiff bases with free amino groups (Patterson & Roberts, 1972). The ability of certain species to metabolize AFB1 into its hemiacetal at higher rates than others constitutes an important aspect of the resistance to the toxin, since the toxicity of AFB2a is much lower than that of the parent compound. For instance, AFB2a has been shown to be not toxic to chicken embryos at levels 100 times the LD50 of AFB1 (Leeson et al., 1995), and the administration of 1.2 mg of AFB2a to one-day-old ducklings does not produce the adverse effects caused by the same dose of AFB1 (Lillehoj & Ciegler, 1969). It has been generally considered that the monohydroxylated metabolites of AFB1 are “detoxified” forms of the toxin, which is probably the case for aflatoxins B2a and Q1; however, AFM1 cannot be considered a detoxication product of AFB1. AFM1 is cytotoxic and carcinogenic in several experimental models and in ducklings its acute toxicity is similar to that of AFB1 (12 and 16 µg/duckling for AFB1 and AFM1, respectively). Also in ducklings, both AFB1 and AFM1 induce similar liver lesions; however, AFB1 induces only mild degenerative changes in the renal convoluted tubules whereas AFM1 causes both degenerative changes and necrosis of the tubules (Purchase, 1967). 2.1.2 O-Demethylation of aflatoxin B1 Another CYP450-mediated reaction of rat, mouse, guinea pig and rabbit livers is the 4-Odemethylation of AFB1. The phenolic product formed was initially isolated from monkey urine (Dalezios et al., 1971) and named aflatoxin P1 (the P comes from the word primate). AFP1 can be hydroxylated at the 9a position to form 4,9a-dihydroxyaflatoxin B1 (AFM1-P1, see Fig. 1), although this compound can also originate from AFM1 (Eaton et al., 1988). AFP1 is generally considered a detoxication product, mainly because it is efficiently conjugated with glucuronic acid (Holeski et al., 1987). There is no evidence that AFP1 or its 9a-hydroxy derivative are produced by any avian species (Leeson et al., 1995).

Biotransformation of Aflatoxin B1 and Its Relationship with the Differential Toxicological Response to Aflatoxin in Commercial Poultry Species

7

2.1.3 Epoxidation of aflatoxin B1 Another metabolic pathway of the vinyl ether double bond present in the AFB1 furofuran ring is its epoxidation. The resultant product, AFB1-exo-8,9-epoxide (AFBO), is an unstable, highly reactive compound, with a half-life of about one second in neutral aqueous buffer (Johnson et al., 1996), that exerts its toxic effects by binding with cellular components, particularly protein, DNA and RNA nucleophilic sites. AFBO is considered to be the active form responsible for the carcinogenicity and mutagenicity of AFB1 (Guengerich et al., 1998). The endo-8,9-epoxide of AFB1 can also be formed by rat and human microsomes (Raney et al., 1992a), but this form of the epoxide is not reactive. Once AFBO is formed it may be hydrolyzed, either catalytically or spontaneously, to form AFB1-8,9-dihydrodiol (AFB1-dhd) or it may be trapped with GSH. If AFB1-dhd is formed it may suffer a base-catalyzed furofuran ring opening to a dialdehyde (AFB1 α-hydroxydialdehyde), which is able to bind to lysine residues in proteins. The enzyme AFAR (see section 2) can protect against the dialdehyde by catalyzing its reduction to a dialcohol which is excreted in the urine either as the dialcohol itself or as a monoalcohol (Guengerich et al., 2001). AFAR activity, however, does not correlate with in vivo sensitivity to AFB1 in selected mammalian models (hamster, mouse, rat and pig) as it was demonstrated by Tulayakul et al. (2005). AFAR has been evidenced by immunoblot in the liver of turkeys (Klein et al., 2002) but its activity has not been investigated in this or any other avian species. 2.1.4 Reduction of aflatoxin B1 The C1 carbonyl group present in the cyclopentanone function of AFB1 can be reduced to a hydroxy group to form the corresponding cyclopentol AFL (Fig. 1). This reaction is not catalyzed by microsomal enzymes but by a cytosolic NADPH-dependent enzyme that in the case of the chicken has an estimated molecular weight of 46.5 KDa and is inhibited by the 17-ketosteroids androsterone, dehydroisoandrosterone and estrone (Chen et al., 1981). Formation of AFL was first reported in chicken, duck, turkey and rabbit liver cytosol (Patterson & Roberts, 1971), and it also occurs in quail (Lozano & Diaz, 2006). However, little or no activity has been observed in guinea pig, mouse or rat liver cytosol (Patterson & Roberts, 1971). AFL can be oxidized back to AFB1 by liver cytosol (Patterson & Roberts, 1972) and by red blood cells from several species (Kumagai et al., 1983). For this reason, AFL is considered to be a "storage" form of AFB1. The ratio of AFB1 reductase activity to AFL dehydrogenase activity in vitro has been observed to be higher in species that are extremely sensitive to acute aflatoxicosis (Wong & Hsieh, 1978), but the significance of this finding in poultry species remains to be determined. AFL cannot be considered a detoxified product of AFB1 since it is carcinogenic and mutagenic, it is acutely toxic to rabbits and it is correlated with susceptibility to AFB1 in some species (Kumagai et al., 1983). Further, AFL has the ability of inducing DNA adduct formation because the double bond between C–8 and C–9 is still present in this metabolite (Loveland et al., 1987). Conjugation of AFL with either glucuronic acid or sulfate would potentially be a true detoxication reaction because this step would prevent AFL from being reconverted to AFB1. 2.1.5 Reduction of aflatoxin B1 metabolites The hydroxylated metabolites AFM1 and AFQ1 can also undergo the cytosolic reduction of the C1 carbonyl group in a reaction analogous to the reduction of AFB1 to AFL. The reduced metabolites of AFM1 and AFQ1 have been named aflatoxicol M1 (Salhab et al., 1977; Loveland et al., 1983) and aflatoxicol H1 (Salhab & Hsieh, 1975), respectively. Aflatoxicol H1

8

Aflatoxins – Biochemistry and Molecular Biology

is a major metabolite of AFB1 produced by human and rhesus monkey livers in vitro (Salhab & Hsieh, 1975). Aflatoxicol M1 can also be produced from AFL and it can be oxidized back to AFM1 by a carbon monoxide-insensitive dehydrogenase activity associated with human liver microsomes (Salhab et al., 1977). 2.2 Phase II metabolism of aflatoxin B1 The most studied Phase II biotransformation reaction of any AFB1 metabolite is the nucleophilic trapping process in which GSH reacts with the electrophilic metabolite AFBO. Conjugation of AFBO with GSH is catalyzed by glutathione transferases (GST, 2.5.1.18), a superfamily of enzymes responsible for a wide range of reactions in which the GSH thiolate anion participates as a nucleophile. These intracellular proteins are found in most aerobic eukaryotes and prokaryotes, and protect cells against chemically-induced toxicity and stress by catalyzing the conjugation of the thiol group of GSH and an electrophilic moiety in the substrate. GSTs are considered the single most important family of enzymes involved in the metabolism of alkylating compounds and are present in most tissues, with high concentrations in the liver, intestine, kidney, testis, adrenal, and lung (Josephy & Mannervik, 2006). The soluble GSTs are subdivided into classes based on sequence similarities, a classification system analogous to that of the CYP450s. The classes are designated by the names of the Greek letters: Alpha, Mu, Pi, and so on, abbreviated in Roman capitals: A, M, P, etc. Within the class, proteins are numbered using Arabic numerals (e.g. GST A1, GST A2, etc.) and specific members are identified by the two monomeric units comprising the enzyme (e.g. GST A1-1, GST A2-2, GST M1-1, etc.). The microsomal GSTs (MGSTs) and its related membrane-bound proteins are structurally different from the soluble GSTs, forming a separate superfamily known as MAPEG (membrane-associated proteins in eicosanoid and GSH metabolism). MGSTs are not involved in the metabolism of AFB1 metabolites. Another conjugation reaction reported for AFB1 metabolites is the conjugation of AFP1 and its 9a-hydroxy metabolite (aflatoxin M1-P1) with glucuronic acid. This conjugation has only been reported in rats and mice (Holeski et al., 1987; Eaton et al., 1988) and leads to the synthesis of detoxified products. Conjugation with glucuronic acid is catalyzed by enzymes known as UPD-glucuronosyltransferases (UGTs, Josephy & Mannevick, 2006), but the specific UGT involved in the conjugation of AFP1 and AFM1-P1 has not been described yet.

3. Biotransformation of aflatoxin B1 in poultry and its relationship with in vivo sensitivity The role of poultry in mycotoxin research in general and aflatoxin research in particular is historically highly relevant since aflatoxins were discovered after a toxic Brazilian peanut meal caused the death of more than 100,000 turkeys of different ages (4-16 weeks) in England during the summer of 1960 (Blount, 1961). This mycotoxicosis outbreak was the first one ever reported for any animal species and for any mycotoxin. Initially only turkeys were affected but later ducklings and pheasants were also killed by the same misterious “X disease“. Interestingly, no chickens were reported to have died from this new disease. Research conducted with poultry after the discovery of aflatoxins (reviewed by Leeson et al., 1995) has clearly shown that the Gallus sp. (which includes the modern commercial meat-type chickens and laying hens) is extremely resistant to aflatoxins while other commercial poultry species are highly sensitive. For instance, whereas ducklings and turkey poults exhibit 100% mortality at dietary levels of 1 ppm (Muller et al., 1970), chicks can tolerate 3 ppm in the diet without showing any observable adverse effects (Diaz & Sugahara, 1995). Interestingly, chickens are

Biotransformation of Aflatoxin B1 and Its Relationship with the Differential Toxicological Response to Aflatoxin in Commercial Poultry Species

9

not only highly resistant to the adverse effects of AFB1 but some studies have reported a modest enhancement in the body weight of chickens exposed to dietary aflatoxins, a finding that has been characterized as an hormetic-type dose-response relationship (Diaz et al., 2008). At the molecular level, at least four mechanisms of action could potentially play a role in the resistance to AFB1: low formation of the putative reactive metabolite (AFBO) and/or AFL, high detoxication of the AFBO and/or AFL formed, intestinal biotransformation of AFB1 before it can reach the liver (“first-pass action”), and increased AFB1 (or toxic metabolites) efflux from the cells. It is important to note that translocation of xenobiotics and their metabolites from the hepatocytes (efflux) mediated by specific basolateral and canalicular transporters (Diaz, 2000) -a process sometimes referred to as Phase III metabolism-, has not been investigated for AFB1 in any species. However, both Phase I and Phase II metabolism appear to have a profound effect on the differential in vivo response to AFB1 in commercial poultry species. The formation of AFBO (by CYP450s) and AFL (by a cytosolic reductase) as well as the scarce information available about detoxication of AFBO through nucleophilic trapping with GSH in poultry will be discussed below. 3.1 Phase I metabolism of aflatoxin B1 in commercial poultry species Research conducted by our group (Lozano & Diaz, 2006) showed that the microsomal and cytosolic biotransformation of AFB1 in chickens, quail, ducks and turkeys results in the formation of two major metabolites: AFBO (microsomes) and AFL (cytosol). The relative in vivo sensitivity to AFB1 in these species corresponds to ducks > turkeys > quail > chicken, and the aim of this work was to try to correlate the toxicological biochemical findings with the reported in vivo sensitivity. Using liver microsomal incubations it was demonstrated that turkeys produce the highest amount of AFBO (detected either as AFB1-dhd or AFB1-GSH) while chickens produce the least; duck and quails produce intermediate amounts (Fig. 2). AFB1 consumption (rate of AFB1 disappearance from the microsomal incubations) was also highest in turkeys, lowest in chickens and intermediate in quail and ducks. Interestingly, these two variables (AFBO production and AFB1 consumption) were highly correlated in the four species evaluated (Fig. 2).

2,5

0,6 2

0,5

1,5

0,4 0,3

1

0,2 0,5

0,1

0

0,0 Turkey

Quail

90,0

3

AFB1-dhd production AFB1 consumption

Duck

Avian species

Chicken

AFB1-dhd production detected as AFB-GSH (nmol AFB-GSH/mg protein/minute)

0,7

AFB1 consumption (nmol AFB1 consumed/mg protein/minute)

AFB1-dhd production (nmol AFB1-dhd/mg protein/minute)

0,8

Turkey

80,0 70,0 60,0 50,0 Quail 40,0

Duck

30,0 Chicken

20,0 10,0 0,0 0,0

0,5 1,0 1,5 2,0 2,5 AFB1 consumption (nmol AFB1 consumed/mg protein/minute)

3,0

Fig. 2. AFBO production (measured as AFB1-dhd) and AFB1 consumption in turkey, quail, duck and chicken microsomal incubations (left) and relationship between AFBO formation (measured as AFB1-GSH) and AFB1 consumption (right).

10

Aflatoxins – Biochemistry and Molecular Biology

Both biotransformation variables (AFBO formation and AFB1 disappearance) correlate well with the in vivo sensitivity observed for turkeys, quail and chickens (turkeys being highly sensitive, chickens being the most resistant and quail having intermediate sensitivity). However, other factor(s) besides AFBO formation and AFB1 consumption must play a role in the extraordinary high sensitivity of ducks to AFB1 because these biochemical variables did not correlate with the in vivo sensitivity for this particular species [ducks exhibit the highest in vivo sensitivity to AFB1 among these poultry species, not turkeys, as Rawal et al. (2010a) affirm]. The cytosolic metabolism of AFB1 in the same four poultry species shows a different trend compared with the microsomal metabolism (Fig. 3). Turkeys are again the largest producers of the cytosolic metabolite AFL but are followed by ducks, chickens and quail (instead of quail, ducks and chickens as it is observed for microsomal metabolism). As discussed before (see section 2.1.4), AFL is a toxic metabolite of AFB1 and it cannot be considered a detoxication product; therefore, it would be expected that sensitive species produce more AFL than resistant ones. However, no correlation between AFL production and in vivo sensitivity was observed. For instance, quail produced the lowest amount of AFL and it exhibits intermediate sensitivity to AFB1, while ducks, which are the most sensitive species, produced much less AFL than turkeys. AFB1 consumption by cytosol (rate of AFB1 disappearance from cytosolic incubations) was highest for the chicken, followed by turkeys, ducks and quail and there was no correlation between AFL formation and AFB1 consumption (Fig. 3). Further, as it was observed for AFL formation, there was no correlation between AFB1 disappearance from cytosol and in vivo sensitivity to AFB1. Investigation of the potential conjugation reactions of AFL might clarify the role of AFL formation on the in vivo sensitivity to AFB1 in poultry. It is possible that the high resistance of chickens to AFB1 might be due to an efficient reduction of AFB1 to AFL followed by conjugation and elimination of the AFL conjugate. Interestingly, it has been demonstrated that chick liver possesses much higher AFB1 reductase activity than duckling or rat liver (Chen et al., 1981).

0,5 0,15

0,4 0,3

0,10

0,2 0,05 0,1 0

0,00 Quail

Duck

Avian species

Chicken

AFL production (nmol AFL/mg protein/minute)

0,6 0,20

Turkey

0,25

0,7

AFL production AFB1 consumption

AFB1 consumption (nmol AFB1 consumed/mg proten/minute)

AFL production (nmol AFL/mg protein/minute)

0,25

Turkey

0,20

0,15 Duck 0,10 Chicken Quail

0,05

0,00 0,0

0,1 0,2 0,3 0,4 0,5 0,6 AFB1 consumption (nmol AFB1 consumed/mg protein/minute)

Fig. 3. AFL production and AFB1 consumption in turkey, quail, duck and chicken cytosolic incubations (left) and relationship between AFL formation and AFB1 consumption (right).

0,7

Biotransformation of Aflatoxin B1 and Its Relationship with the Differential Toxicological Response to Aflatoxin in Commercial Poultry Species

11

Even though the studies of Klein et al. (2000) in turkeys, and Lozano & Diaz (2006) in turkeys, chickens, ducks and quail had clearly demonstrated that hepatic microsomes from poultry were capable of bioactivating AFB1 into AFBO, there was only scarce information on the specific CYP450 enzymes responsible for this biotransformation reaction and it was limited to turkeys (Klein et al., 2000; Yip & Coulombe, 2006). In contrast, in humans, at least three CYP450s had been identified as responsible for AFB1 bioactivation to AFBO (CYP1A2, CYP2A6 and CYP3A4) (Omiecinski et al., 1999; Hasler et al., 1999), and there was evidence that the CYP3A4 human enzyme was the most efficient (Guengerich & Shimada, 1998). In view of this lack of information a series of studies were conducted by our group (Diaz et al., 2010a, 2010b, 2010c) in order to investigate which specific avian CYP450 orthologs were responsible for the bioactivation of AFB1 into AFBO. These studies were conducted by using specific human CYP450 inhibitors (αnaphthoflavone for CYP1A1/2, furafylline for CYP1A2, 8-methoxypsoralen for CYP2A6 and troleandomycin for CYP3A4), by correlating AFBO formation with human prototype substrate activity (ethoxyresorufin O-deethylation for CYP1A1/2, methoxyresorufin Odeethylation for CYP1A2, coumarin 7-hydroxylation for CYP2A6 and nifedipine oxidation for CYP3A4) and by investigating the presence of ortholog proteins in avian liver by immunoblot using antibodies specific against human CYP1A1, CYP1A2, CYP2A6 and CYP3A4. These series of studies revealed that the avian CYP2A6 ortholog is the main CYP450 enzyme responsible for the bioactivation of AFB1 into its epoxide form in all poultry species investigated. Evidences for this conclusion include the fact that AFBO production was inhibited by the CYP2A6 inhibitor 8-methoxypsoralen and that a significant correlation existed between coumarin 7-hydroxylation and AFB1 epoxidation activity in all species studied (Table 1). The finding of a protein by immunoblot using rabbit anti-human CYP450 polyclonal antibodies directed against the human CYP2A6 enzyme confirmed the existence of an immunoreactive protein in all birds studied (the putative CYP2A6 avian ortholog). These studies demonstrated for the first time the existence of the CYP2A6 human ortholog in avian species and they were the first reporting the role of this enzyme in AFB1 bioactivation in avian liver. Poultry Species Turkey Quail Duck Chicken

7-Ethoxyresorufin- 7-MethoxyresorufinO-demethylation O-demethylation (CYP1A1/2) (CYP1A2) 0.32 -0.76 -0.09 0.21 0.81 0.82 0.25 0.46

Coumarin 7hydroxilation (CYP2A6) 0.90 0.78 0.68 0.83

Nifedipine oxidation (CYP3A4) 0.73 0.07 0.88 -0.24

Table 1. Pearson correlation coefficients for aflatoxin B1 epoxidation vs. prototype substrate activities of selected human CYP450 enzymes. Correlations in bold numbers are statistically significant (P ≤ 0.01). In turkeys, quail and chickens the CYP1A1 ortholog seems to have a minor role in AFB1 bioactivation, while in ducks there are evidences that AFB1 bioactivation is carried out not only by the CYP2A6 and CYP1A1 orthologs but also by the CYP3A4 and CYP1A2. The fact that four CYP450 enzymes are involved in AFB1 bioactivation in ducks could partially explain the high sensitivity of this species to AFB1. In turkey liver, AFB1 was

12

Aflatoxins – Biochemistry and Molecular Biology

reported to be activated to AFBO by a CYP 1A ortholog (Klein et al., 2000) that later was identified as the turkey CYP1A5 on the basis of its 94.7% sequence identity to the CYP1A5 from chicken liver (Yip & Coulombe, 2006). This enzyme was suggested to correspond to the human ortholog CYP1A2 (Yip & Coulombe, 2006). However, using human prototype substrates and inhibitors, Diaz et al. (2010a) found evidence for AFB1 bioactivation by CYP1A1 but not by CYP1A2 in turkey liver microsomes. Interestingly, the turkey CYP1A5 has a high amino acid sequence homology not only with the human CYP1A2 (62%) but also with the human CYP1A1 (61%) as reported by the UniProtKB database (http://www.uniprot.org) and the National Center for Biotechnology Information database (http://www.ncbi.nlm.nih.gov). It is possible that the turkey CYP1A5 enzyme cloned by Yip & Coulombe (2006) may in fact correspond to the human CYP1A1 ortholog or, even more interesting, to both the CYP1A1 and 1A2 human orthologs. Murcia et al. (2011) found a very high correlation between EROD (CYP1A1/2) and MROD (CYP1A2) activities in turkey liver microsomes (r=0.88, P turkey poults > goslings > pheasant chicks > chickens (Muller et al., 1970). Ducklings are 5 to 15 times more sensitive to the effects of aflatoxins than are laying hens, but when laying hen strains are compared, certain strains of hens may be as much as 3 times more sensitive than other strains (Jones et al., 1994). In comparing sensitivity of different strains of leghorn chicks (Table 5), it was found there is up to a 2.5 difference in the LD50 dose at 6 weeks of age (FDA, 1979) Strain A B C D E F

LD50 mg/kg 6.5 7.25 9.25 9.50 11.50 16.50

Table 5. Sensitivity in different leghorn strains of chicks In poultry, aflatoxin impairs all important production parameters including weight gain, feed intake, feed conversion efficiency, pigmentation, processing yield, egg production, male and female reproductive performance. Some influences are direct effects of intoxication, while others are indirect, such as from reduced feed intake (Calnek et al., 1997). The direct and indirect effects of aflatoxicosis include increased mortality from heat stress (broiler breeders, Dafalla et al., 1987a), decreased egg production in leghorns, (Bryden et al., 1980), anemia, hemorrhages and liver condemnations (Lamont, 1979), paralysis and lameness (Okoye et al., 1988), impaired performance in broilers, (Jones et al., 1982), increased mortality rate in ducks, (Bryden et al., 1980), impaired ambulation and paralysis in quail, (Wilson et al., 1975), impaired immunization in turkeys, (Hegazy et al., 1991), and increased susceptibility to infectious diseases (Bryden et al., 1980 and Calnek et al., 1997).

Aflatoxins and Aflatoxicosis in Human and Animals

237

2.11.1 Chickens Susceptibility of chickens to toxic effects of AFB1 varies with several factors such as breed, strain, age, nutritional status, amount of toxin intake and also the capacity of liver microsomal enzymes to detoxify AFB1 (Edds, 1973; Veltmann, 1984). Acute toxicity of aflatoxins in chickens may be characterized by hemorrhage in many tissues and liver necrosis with icterus. Although number of field cases of aflatoxicosis in chickens has been diagnosed in various countries, the most severe spontaneous outbreak occurred in North Carolina, in which 50% of a flock of laying hens died within 48 hr of being fed highly toxic maize containing 100 ppm aflatoxin (Hamilton, 1971). The necropsy revealed that liver damage was the most important lesion showing paleness, occasional white pinhead-sized foci and petechial hemorrhages while gallbladder and bile ducts were distended. Levels of aflatoxin B1 in moldy feed normally vary from 0 to 10 ppm. At low levels of feed contamination, exposed chickens show, in general, weakness, failure to gain weight with concomitant decline in feed efficiency and egg production (Smith and Hamilton, 1970; Doerr et al.,1983). Hepatic damage is manifested by enlarged and putty-colored liver, petechial hemorrhages, marked vacuolation of hepatic cells and bile duct proliferation. Feed levels of AFB1 as low as 250-500 ppb given to New Hampshire chickens have been reported to result in liver damage, decreased hemoglobin, and hypoproteinemia (Brown and Abrams, 1965). Experimental trials with naturally contaminated feed containing aflatoxin levels ranging from 1-1.5 ppm have caused growth retardation in chickens. Mortality was low but marked hepatic damage was manifested by enlarged and hemorrhagic liver (Carnaghan et al., 1966). Relatively, high dietary levels of aflatoxin B1 (0-10 ppm) given to Rock type broiler chickens have been reported to cause substantial decrease in weight gain, feed efficiency and hepatic microsomal drug metabolizing enzymes with concomitant increase in serum glutamic oxalacetic transaminase activity reflecting liver damage (Dalvi and McGowan, 1984; Dalvi and Ademoyero, 1984). Metabolic alterations caused by aflatoxins in chickens result in elevated lipid levels(Tung et al., 1972; Donaldson et al., 1972), disruptions in hepatic protein synthesis (Tung et al.,1975) which result in several blood coagulation disorders (Doerr et al.,1976; Bababunmi and Bassir, 1982), immunosuppression and decreased plasma amino acid concentrations (Voight et al., 1980). 2.11.2 Ducks Lethal aflatoxicosis in ducklings occurred as inappetance, reduced growth, abnormal vocalizations, feather picking, purple discoloration of legs and feet and lameness. Ataxia, convulsions and opisthotonus preceded death (Asplin and Carnaghan, 1961). At necropsy, livers and kidneys were enlarged and pale. With chronicity, ascitis and hydropericardium developed accompanied by shrunken firm nodular liver, distention of the gall bladder and hemorrhages (Asplin and Carnagham, 1961; Calnek et al., 1997), distended abdomen due to liver tumors and secondary ascitis (Hetzel et al., 1984). Microscopic lesions in the liver were fatty change in hepatocytes, proliferation of bile ductules and extensive fibrosis accompanied by vascular and degenerative lesions in pancreas and kidney (Asplin and Carnagham, 1961 and Calnek et al., 1997). Bile duct hyperplasia and bile duct carcinoma are also reported (Hetzel et al., 1984) in aflatoxicated Campbell ducks.

238

Aflatoxins – Biochemistry and Molecular Biology

2.11.3 Turkeys The initial clinical signs reported during the outbreak of Turkey “x" disease were anorexia and weight loss followed by depression, ataxia and recumbency. Affected birds died with in a week or two and at the time of death frequency had opisthotonus characterized by arched neck, head down back and legs extended backwards (Hamilton et al., 1972). Along with decreased feed conversion and weight gain, reduced spontaneous activity, unsteady gait, recumbency, anemia and death (Siller and Ostler, 1961; Wannop, 1961; Giambrone et al., 1985 ; Richard et al., 1987). At necropsy, the body condition was generally good but there was generalized congestion and edema. The liver and kidney were congested, enlarged and firm, the gall bladder was full, and the duodenum was distended with catarrhal content (Siller and Ostler, 1961; Wannop, 1961; Calnek et al., 1997). 2.11.4 Broilers Decreased water and feed intake, weight loss, dullness, stunting, ruffled feathers, poor appearance and paleness, trembling, ataxia, lameness, paralysis of the legs and wings gasping, prostration and death are frequency seen in experimental and natural outbreak of aflatoxicosis in broilers (Asuzu and Shetty, 1986; Okoye et al., 1988; Rao and Joshi, 1993 ; Leeson et al., 1995). The most characteristic gross lesions appeared in the livers which were enlarged, pale yellow to grayish brown and had a prominent reticular pattern. Petecheal hemorrhages were observed on the surface of some livers. Gall bladders were enlarged and bile duct distended and there were blood in the intestinal lumen (Archibald et al., 1962; Azuzu and Shetty, 1986). The liver, spleen and kidney were increased in size, whereas the bursa of fabricius and thymus were decreased (Smith and Hamilton, 1970; Huff and Doerr, 1980). Lethal aflatoxicosis can cause either dark red or yellow discoloration of the liver due to congestion or fat accumulation, respectively (Slowik et al., 1985). At chronicity livers became shrunken, firm and nodular and gall bladder was distended (Asplin and Carnaghan, 1961). The kidneys of affected birds appeared enlarged and congested (Tung et al., 1973) and the spleen will be enlarged and mottled in appearance (Tung et al., 1975 a). Histopathology of the liver revealed congestion of hepatic sinusoids, fecal hemorrhages, centro-lobular fatty cytoplasmic vacuolation and necrosis, biliary hyperplasia and nodular lymphoid infiltration. In the kidney, the epithelial cells of many tubules were vacuolated (Dafalla et al., 1987 b). Azuza and Shetty (1986) and Okoye et al., (1988) observed severe degeneration of hepatocytes, dilation of central veins, bile duct proliferation and lymphocytic depletion in lymphoid organs in field outbreaks of aflatoxicosis in broilers. 2.11.5 Laying hens Reduced egg production and egg weight, enlarged liver and increased liver fat are the most prominent manifestations of experimental aflatoxicosis in layers (Nesheim and Lvy, 1971; Hamilton and Garlich, 1973; Leeson et al., 1995). High mortality and dramatic reduction of egg production were reported to occur during a natural outbreak (Hamilton, 1971). Egg size, egg weight and yolk as percent of total egg size are decreased (Huff et al., 1975). In Japanese quail, decreased feed conversion, egg production, egg weight, hatchability and exterior and interior egg quality were detected (Sawhney et al., 1973a & b). Dhanasekaran et al., (2009) reported that histopathological analysis of aflatoxin ingested hens reveals that lesions were

239

Aflatoxins and Aflatoxicosis in Human and Animals

observed in tissues of liver, kidney, intestine (Plate 1). Jayabharathi and Mohamudha parveen (2010) tested the aflatoxicosis in hens. Haematological analysis showed the decreased haemoglobin than the control group (Plate2).

Control Hens with various organs

Test Hens with accumulation of fatty layer

Lung (Control)

Lung (Test)

Plate 1. Organal view of Hens with Aflatoxicosis

Normal lung cells (Control)

Mild infiltration by L and P (Test)

240

Aflatoxins – Biochemistry and Molecular Biology

Dense infiltration by L and P (Test)

Infiltration by granular structures suspicious of carcinoma

Normal Intestinal mucosa (Control)

Atypical granular structures some with enlarged nuclei (Test)

Stomach cells (Control)

Infiltration of glandular structure (Test)

Renal tissue with abnormalities (Test)

Intestine mildly dilated glandular structure (Test)

Plate 2. Histopathological analysis of various organs of hens with aflotoxicosis

Aflatoxins and Aflatoxicosis in Human and Animals

241

2.12 Reproduction and hatchability Aflatoxins causes delayed maturation of both males and females (Doerr, 1979; Doerr and Ottinger, 1980). Aflatoxicosis in white leghorn males resulted in decreased feed consumption, body weight, testes weight and semen volume (Sharlin et al., 1980), and decreased plasma testosterone values (Sharlin et al., 1980). While in broiler breeder males reduction in body weight and mild anemia with no alterations in semen characteristics were observed (Wyatt, 1991; Briggs et al., 1974). In mature laying hens experiencing aflatoxicosis, enlarged and fatty liver and marked decrease in egg production were observed (Hamilton and Garlich, 1972). Severe decline in hatchability was recorded in mature broiler breeder hens after consumption of aflatoxin (Howarth and Wyatt, 1976). Hatchability declines before egg production and is the most sensitive parameter of aflatoxicosis in broiler breeder hens (Howarth and Wyatt, 1976). The immediate and severe decline in hatchability was found to arise from an increase in early embryonic mortality rather than infertility of the hens. The cause of the increased embryonic mortality is the transfer of toxic metabolites from the diet of the hen to the egg (Wyatt, 1991). The delayed response in egg production is thought to occur due to reducing synthesis and transport of yolk precursors in the liver (Huff et al., 1975). 2.13 Immunosuppression Aflatoxin induces immunosuppression and increases susceptibility of toxicated birds to bacterial, viral and parasitic infections. Immunosuppression caused by AFB1 has been demonstrated in chickens and turkeys as well as laboratory animals (Sharma, 1993). Aflatoxin decreases the concentrations of immunoglobulins IgM, IgG and IgA in birds (Giambrone et al., 1978a & b). The presence of low levels of AFB1 in the feed appears to decrease vaccinal immunity and may therefore lead to the occurrence of disease even in properly vaccinated flocks (Leeson et al., 1995). Thaxton et al., (1974) recorded reduced antibody production following injection of sheep red blood cells in chickens experiencing aflatoxicosis. Batra et al., (1991) found that chickens fed AFB1 and vaccinated against Marek's disease showed a significantly higher frequency of gross and microscopical lesions of Marek's disease than did chickens fed aflatoxin-free diet. Cell-mediated immune response and effector cell function are also affected during aflatoxicosis (Leeson et al.,1995). Aflatoxin decrease complement activity in chickens (Campbell et al., 1983 and Stewart et al., 1985) and turkeys (Corrier, 1991). Since complement is required for normal phagocytosis, impairment in complement activity may partially explain impairment of phagocytosis in chickens experiencing aflatoxicosis (Gewurz and Suyehira, 1976; Wyatt, 1991). Chang and Hamilton (1979a) demonstrated reduced chemotactic ability of leucocytes, impaired phagocytosis of heterophils and impaired cellular and serum factors required for optimal phayocytosis in aflatoxicated chickens. Although thrombocytic counts are depressed by dietary aflatoxin (Mohiuddin et al., 1986) their phagocytic activity is not affected by aflatoxin (Chang and Hamilton, 1979b). However, other phagocytic cells (heterophils, macrophages and monocytes) were affected by dietary aflatoxin (Chang and Hamilton, 1979a). Chickens receiving aflatoxin-contaminated diets showed higher susceptibility to Marek's disease (Edds and Bortell, 1983), infectious bursal disease virus (Giambrone et al., 1978a & b), congenitally acquired salmonellosis (Wyatt and Hamilton, 1975) and duodenal and cecal coccidiosis (Edds et al., 1973) than chickens receiving aflatoxin free diet.

242

Aflatoxins – Biochemistry and Molecular Biology

From the aforementioned, it is postulated that aflatoxin interferes with normal function of B and T lymphocytes, rather than causing destruction of these cells (Wyatt, 1991). The impairment of protein synthesis caused by dietary aflatoxin could account for the lack of humoral immunity without the necessity of B and T cell destruction (Wyatt, 1991). Regardless the atrophy of the bursa of fabricius and thymus gland, the apparent alteration of splenic function is also of diagnostic significance and implies alteration in the immunocompetence of birds with aflatoxicosis (Richard et al., 1975). 2.14 Hematological and biochemical alterations Aflatoxin causes hematopoietic suppression and anemia observed as decreases in total erythrocytes, packed-cell volume and hemoglobin (Reddy et al.,1984; Huff et al., 1986; Mohiuddin et al., 1986). Total leucocytes are increased and differential leucocytic counts vary among studies with concurrent lymphopenia (Tung et al., 1975a; Lanza et al., 1980), monocytoses and heterophilia (Wannop, 1961). Aflatoxin is known to produce hemolytic anemia by decreasing the circulating mature erythrocytes. Lysis of erythrocytes will result in above the normal levels of cellular debris in circulation (Tung et al., 1975a) and consequently the spleen appear congested because of an unusually high concentration of inorganic iron and debris from the circulation (Wyatt, 1991). Several biochemical parameters are affected by aflatoxin exposure. Aflatoxin decreases total serum proteins, alpha, beta and gamma globulins, with IgG being more sensitive than IgM (Tung et al., 1975a). Total serum proteins contents are depressed due to reduced values of alpha and beta globulins and albumen, while gamma globulins are affected more variably (Pier, 1973). Serum lipoproteins, cholesterol, triglycerides, uric acid and calcium are also decreased (Garlich et al., 1973; Doerr et al., 1983; Reddy et al.,1984; Huff et al., 1986). The activity of serum or plasma enzymes has been extensively used as a measure of aflatoxin activity in chickens. Increased activities of sorbitol dehydrogenase, glutamic dehydrogenase, lactate dehydrogenase, alkaline phosphatase, acid phosphatase, aspartate aminotransferase and alanine aminotransferase were reported in aflatoxicated chickens (Dafalla et al., 1987b; Rao and Joshi, 1993; Leeson et al., 1995). The increase in the levels of serum enzymes measured was interpreted as a consequence of hepatocyte degeneration and subsequent leakage of enzymes (Leeson et al., 1995). Aflatoxin has also shown to alter both the extrinsic and common clotting pathways in chickens. Aflatoxins causes biochemical changes in thromboplastin clotting factors V, VII and X and reduces plasma prothrombin and fibrinogen (Doerr et al., 1976), and consequently increases whole blood clotting and prothrombin times (Doerr et al., 1974). The elevated prothrombin time was considered to be the result of impaired hepatic synthesis of clotting factors caused by the toxication of aflatoxin on the liver cells (Huff et al., 1983). The activity of some digestive enzymes, the absorption of carotenoid compounds from the gastrointestinal tract, and the metabolism of lipids can be altered by aflatoxin exposure (Leeson et al., 1995). Dietary aflatoxin produced a malabsorption syndrome characterized by steatorrhea, hypocarotenoidemia and decreased concentrations of bile salts and pancreatic lipase, trypsin, amylase and Rnase (Osbrone et al.,1982). In another experiment, the specific activities of pancreatic chymotrypsin, amylase and lipase, but not trypsin were increased significantly by aflatoxin (Richardson and Hamilton, 1987).

Aflatoxins and Aflatoxicosis in Human and Animals

243

The effect of aflatoxin on the renal function of broiler chickens was reported by Glahn (1993). Aflatoxin treated birds showed decreased fractional excretion of phosphate, total plasma calcium concentration, decreased total plasma proteins, plasma 25-hydroxyl vitamin D and plasma 1, 25-dihydroxy vitamin D. 2.15 Wild life Birds, fishes and mammals vary among species in susceptibility to aflatoxins. Birds such as bobwhite quail and wild turkey appear to be more susceptible than mammals (Horn et al., 1989). It is difficult to document the extent to which wildlife species are affected because wild animals are free roaming and elusive. In many cases, predators and scavengers may consume dead or dying animals before the dead animals are found by humans (Stewart and Larson, 2002). Clinical signs of aflatoxicosis in wildlife vary according to the dose received, the time period of exposure, and species of animal. Toxic effects can be divided into acute, subacute and chronic exposures (Stewart and Larson, 2002). Acute effects reflect severe liver disease. Animals may be anemic and may exhibit difficulty in breathing. Sudden death with no clinical signs may occur. Subacute effects may allow animals to live for a longer period of time. These animals have yellow eyes, mucous membranes, or yellowed skin along with abnormalities in blood clotting. Bruising, nose bleeds and hemorrhaging may be observed. Chronic effects are generally related to impaired liver function. Long-term, low-level consumption of aflatoxins may result in reduced feed efficiency, weight loss, lack of as appetite and increased receptivity to secondary infectious diseases. Lesions may occur in the liver and other organs and fluid may accumulate in the body cavity. 2.15.1 Fish Fish have been found susceptible to aflatoxin and trichothecenes. Aflatoxicosis is most prevalent among fishes. The extent of lesions caused by consumption of aflatoxins depends upon the age and species of the fish. Fry are more susceptible to aflatoxicosis than adults and some species of fish are more sensitive to aflatoxins than others (Royes and Yanong, 2002). Rainbow trout are the most sensitive species to aflatoxin. Feeding trout diets containing less than 1 ppb will cause liver tumors in 20 months. (Horn et al., 1989). Diet containing AFB1 at 0.4 ppb for 15 months had a 14% chance of developing tumors. Feeding trout a diet containing 20 ppb for 8 months resulted in 58% occurrence of liver tumors and continued feeding for 12 months resulted in 83% incidence of tumors (Royes and Yanong, 2002). Deaths quickly occur in 50% of stock if dietary levels of 500 to 1000 ppb are consumed. Warm water fishes such as channel catfish (Ictalurs punctatus) are much less sensitive than rainbow trout, and the level required to cause 50% mortality is approximately 30 times that of rainbow trout (Horn et al., 1989). Channel catfish fed a diet containing purified AFB1 at 10.000 ppb for 10 weeks exhibited decreased growth rate and moderate internal lesions (Royes and Yanong, 2002). Initial findings associated with aflatoxicosis in fishes include pale gills, impaired blood clotting, poor growth rates or lack of weight gain. Prolonged feeding of low concentrations of AFB1 causes liver tumors, which appear as pale yellow lesions and which can spread to the kidney. Increased in mortality may be observed (Royes and Yanong, 2002).

244

Aflatoxins – Biochemistry and Molecular Biology

Aflatoxin can also depress the immune system indirect through their effect on enextial nutrients in the diet, making fish more susceptible to bacterial, viral and parasitic diseases. Moreover, aflatoxin can cause slow growth rate and reduced weight of the finished product of warm-water fish (Royes and Yanong, 2002).

3. Conclusion This chapter describes the food sources of aflatoxin contamination and their diseases in human and animals such as cattles, poultry, fish and other wild animals. Quality of food and feed plays the most important role in the farming as its share is 70%. Good quality food and resistant strain of animals can lead to greater production and more profit for the poultry, dairy, fishery former. However, the acute shortage of chicken, mutton, fish meat has pushed its prices steeply upwards. It is suggested that use of chicks, cow, sheep, fish, dog, horses are resistant to aflatoxicosis, would help in minimizing problem of poor growth rate and poor feed conversion which perhaps are the two most important factors in animal management.

4. References Abdelsalam, E. B.; Eltayeb, A.F.; Noreidin, A.A. & Abdulmagid, A. M.(1989). Aflatoxicosis in fattening sheep. Vet. Rec, Vol. 124, pp. 487-488. Agag, B.I. (2004). Mycotoxins in foods and feeds 1-Aflatoxins. Ass Univ. Bull. Environ. Res, Vol.7, No.1, pp.173-205 Allcroft, R.; Roberts, B. A. & Lloyd, M. K. (1968). Excretion of aflatoxin in a lactating cow. Food Cosmet. Toxicol, Vol.6, pp.619- 625 Andrellos, P. J. & Reid, G.B (1964). Confirmatory tests for aflatoxin B. J. Assoc. Offic. Ag, Chemists Vol.47, pp.801-803. Angsubhakorn, S.; Poomvises, P. Romruen, K. & Newbern, P.M. (1981). Aflatoxicosis in horses. Am, Vet. Med. Asso,. Vol.178, pp.274-278. Applebaum, R.S.; Brackett, R.E., Wiseman, D.W., & Marth, E.H. (1982). Aflatoxin toxicity to dairy cattle and occurrence in milk and milk products. J. Food. Pro, Vol.45, No.8, pp.752-777. Archibald, R.; Smith, H.J. & Smith, J.D. (1962). Brazilian groundnut toxicosis in Canadian broiler chickens. Can. Vet. J, Vol.3. pp.322-325. Armbrecht, B.H.; Shalkop, W.T. Rollins, L.D. Pohland, A.E. & Stoloff, L. (1970). Acute toxicity of AFB1 in weathers. Nature, Lond. Vol.225, pp.1062-1063 Asao, T. G.; Buchi, M. M, Abdel kader, S. B, Chang, E. L, Wick, & Wogan G.N. (1963). Aflatoxins B and G. J. Am. Chem. So, Vol.85, pp.1706-1707 Asplin, F.D.& Carnaghan, R.B. (1961). The toxicity of certain groundnut meals for poultry with special reference to their effect on ducklings and chickens. Vet. Res, Vol.3, pp. 1215-1219. Asuzu, I.U. & Shetty, S.N. (1986). Acute aflatoxicosis in broiler chicken in Nsuka, Nigeria. Trop. Vet, Vol.4, pp.79-80 Bababunmi, E. A.& Bassir, O. (1969). The effect of aflatoxin on blood clotting in the rat. Brit. J. Pharmacol, Vol.37, pp.497-500

Aflatoxins and Aflatoxicosis in Human and Animals

245

Bassir, O. & Osiyemi, F. (1967). Biliary excretion of aflatoxin in the rat after a single dose. Nature, Vol.215, pp.882 Bastianello, S.S.; Nesbit, J.W. Willians, M.C. & Lange, A.L. (1987). Pathological findings in a natural outbreak of aflatoxicosis in dogs. Ondersteport J. Vet. Res, Vol.54, pp.635640. Batra, P.; Pruthi, A.K. & Sadana, J.R. (1991). Effect of AFB1 of the efficacy of turkey herpes virus vaccine against Marek's disease. Res. Vet. Sc, Vol.51, pp.115-119 Blaney, B.K. & Williams, K.C. (1991). Effective use in livestock feeds of mouldy and weather damaged grain containing mycotoxins. Case histories and economic assessments pertaining to pig and poultry industries of Queensland. Aust. J. Agric. Res,Vol.42, pp.993-1012 Bodine, A.B. & Mrtens, D.R. (1983). Toxicology, metabolism and physiological effects of aflatoxin in the bovine. pp.46-50. In: U.L. Diener; R.L. Asquith and J.W. Dickens Eds. Aflatoxin and A. flavus in corn. AAES, Auburn Univ., Alabama. Bortell, R.; Asquith, R.L. Edds, G.T. Simpson, C.F. Aller, W.W. (1983). Acute experimentally induced aflatoxicosis in the weanling pony. American Journal of Veterinary Research, Vol.44, pp.2110–2114 Brackett, R.E. & Marth, E.H. (1982). Association of AFM1 with casein.Z. Lebensm Unters Forsch, Vol.174, pp.439 Briggs, D.M.; Wyatt, R.D.& Hamilton, P.B. (1974).The effect of dietary aflatoxin on semen characteristics of mature broiler breeder males. Poult. Sci, Vol.53, pp.2115-2119 Brown, J.M.M. & Abrams, L. (1965). Biochemical studies on aflatoxicosis. Onderstepoort, J. Vet. Res., Vol. 32, pp. 119-146. Bryden, W.L.; Lioyd, A.B. & Cumming, R.B. (1980). Aflatoxin contamination of Australian animal feeds and suspected cases of mycotoxicosis. Aust. Vet. J, Vol.56, pp.176-180 Buchi, G.; Spitzner, D. Paglialunga, S. & Wogan, G. N. (1973). Synthesis and toxicity evaluation of aflatoxin P1. Life Sci, Vol .3, pp.1143-1149 Burg, W.R.& Shotwell, O.L. (1984). Aflatoxin levels in airborne dust generated from contaminated corn during harvest and at an elevator in 1980. Journal of the Association of Official Analytical Chemists Vol.67, pp.309–312 Burnside, J.E.; Sipple, W.L. Forgacs, J, Carll, W.T, Atwood, M.B. & Doll, E.R. (1957). A disease of swine and cattle caused by eating moldy corn. II. Experimental production with pure cultures of molds. Am. J. Vet. Res, Vol.18, pp.817 Calnek, B.C.; Barnes, H.J. MCDougald, L.R. & Saif, Y.M. (1997). Diseases of poultry. 10th ed., pp.951-979. Mosby-Wolfe, Iowa state Univ. press, Ames, Iowa, USA. Caloni, F. & Cortinovis.C (2011). Toxicological effects of aflatoxins in horses. The veterinary journal. Vol.188, pp. 270-273 Campbell, T.C. & Hayes, J.R. (1976). Toxicol. Appl. Pharmacol, Vol.35, pp.199-222. Cited by Hsieh (1983). Carnaghan, R. B. A.; Lewis, G. Patterson, D. S. P. & Allcroft R. (1966). Biochemical and pathological aspects of groundnut poisoning in chickens. Pathol. Vet.3, pp.601-615 Cassel, E.K.; Barao, S.M. and Carmal, D.K. (1988). Aflatoxicosis and ruminants. Texas Vet. Med. Diagnostic lab, Tesas college. The national dairy database (1992) NDB, Health, Test, Hf100200. TxT

246

Aflatoxins – Biochemistry and Molecular Biology

CAST, Council for Agric. Sci. and Technol. (1989). Mycotoxins economic and health risks. Task force report No.116 Chaffee, V.W.; Edds, G.T. Himes, J.A. & Neal, F.C. (1969). Aflatoxicosis in dogs. Am. J. Vet. Re.,Vol.30, pp.1737-1749 Chang, C.F. & Hamilton, P.B. (1979a). Impaired phagocytosis by heterophils from chickens during aflatoxicosis. Toxicol. Appl. Pharmacol, vol.6, pp.459 Chang, C.F. & Hamilton, P.B. (1979b). Refractory phagocytosis by chicken thrombocytes during aflatoxicosis. Poult. Sci, Vol.58, pp.559-561 Colvin, B.M.; Harrison, L.R. Grosser, H.S. & Hall, R.F. (1984). Aflatoxicosis in feeder cattle. J. Am. Vet. Med. Assoc.Vol.184. pp.956. Cook, W.O.; Richard, J.L. Osweiller, G.D. & Trampel, D.W. (1986). Clinical and pathological changes in acute bovine aflatoxicosis. Rumen motility and tissue and fluid concentrations of aflatoxins B1 and M1. Am. J. Vet. Res, Vol.47, pp.1817-1825 Cook, W.R. ( 1976). Chronic bronchitis and alveolar emphysema in the horse. Veterinary Record,Vol.99, pp.448–451 Corrier, D.E. (1991). Mycotoxicosis: Mechanism of immunosuppression. Vet. Immunol. Immuno Pathol, Vol.30, pp.73-87 Cysewski, S.J.; Pier, A.C. Baetz, A.L.& Cheville, N.F. (1982). Experimental equine aflatoxicosis. Toxicology and Applied Pharmacology, Vol.65, pp. 354–365 Dafalla, R.; Hassan, Y.M. & Adam, S.E.I. (1987a). Fatty and hemorrhagic liver and kidney syndrome in breeding hens caused by AFB1 and heat stress in the Sudan. Vet. Hum. Toxicol, Vol.29, pp.222-226 Dafalla, R.; Yagi, A.I. & Adam, S.E.I. (1987b). Experimental aflatoxicosis in Hybro-type chicks: Sequential changes in growth and serum constituents and histopathological changes. Vet. Human Toxicol,Vol.29, pp.222-225 Dalezios, J. I.; Wogan, G. N. & Weinhreb, S. M. (1971). Aflatoxin P1: A new aflatoxin metabolite in monkeys. Science ,Vol.171, pp.584-585 Dalvi, R.R. & McGowan, C. (1984). Experimental induction of chronic aflatoxicosis in chickens by purified aflatoxins B1 and its reversal by activated charcoal, Phenobarbital and reduced glutathione. Poult. Sci, Vol. 63, PP. 485-491. Dalvi, R.R. & Ademoyero,A.A. (1984). Toxic effects of aflatoxins B1 in chickens given feed contaminated with aspergillus flavus and reduction of the toxicity by activated charcoal and some chemical agents. Avia. dis, Vol. 28, PP. 61-69. De Iongh, H.; Beerthuls, R. K. Vles, R. 0. Barrett, C. B. & Ord, W. 0. (1962). Investigation of the factor in groundnut meal responsible for Turkey “x” disease. Biochim. Biophys. Acta, Vol.65, pp.548-551 DeIongh, H., Vles, R. O. & VanPelt, J. G. (1964). Milk of mammals fed an aflatoxin containing diet. Nature, Vol. 202, pp.466-467 Devero, A. (1999). Aflatoxins: The effects on human and animal health. Biol, 4900; Fall, 1999 Dhanasekaran, D.; Panneerselvam, A. & Thajuddin,N. (2009). Evaluation of Aflatoxicosis in hens fed with commercial poultry feed. Turk. J. Vet. Anim. Sci, Vol.33, No.5. pp.385391 Diekman, A.; Coffey, M.T. Purkhiser, E.D. Reeves, D.E. & Young, L.G. (1992). Mycotoxins and swine performance. CES, PTH-129, Purdue Univ., West Lafayette, Indian.

Aflatoxins and Aflatoxicosis in Human and Animals

247

Diener, U.L.& Davis, N.D. (1968): "Effect of environment on aflatoxin production in peanuts. Tropical science, Vol.10, pp.22-25 Doerr, J.A. & Ottinger, M.A. (1980). Delayed reproductive development resulting from aflatoxicosis in Juvenile Japanese quail. Poult. Sci, Vol.59, pp.1995-2001 Doerr, J.A. & Ottinger, M.A. (1980). Delayed reproductive development resulting from aflatoxicosis in Juvenile Japanese quail. Poult. Sci., Vol.59, 1995- 2001. Doerr, J.A. (1979). Mycotoxicosis and avian hematosis. Diss. Abstr. B Sci. Eng., 4127. Doerr, J.A.; Huff, W.E. Wabeck, C.J. Chaloupka, G.W. May, J.D. & Merkley, J.W. (1983). Effects of low level chronic aflatoxicosis in broiler chickens. Poult. Sci,Vol.62, pp.1971-1977 Doerr, J.A.; Huff, W.E. Wyatt, R.D. & Hamilton, P.B. (1974). Survey of T-2 toxin, ochratoxin and aflatoxin for their effects on the coagulation of blood in young broiler chickens. Poult. Sci, Vol.53, pp.1729 Doerr, J.A.; Wyatt, R.D. & Hamilton, P.B. (1976). Impairment of coagulation function during aflatoxicosis in young chickens. Toxicol. Appl. Phormacol, Vol.35, pp.437 Donaldson, W, E.; Tung, H.T. & Hamilton, P.B. (1072). Depression of fatty acid synthesis in chick liver (Gallus domesticus) by aflatoxins. Comp. Biochem. Physiol, Vol.41, pp. 843847. Eaton, D.L. and Groopman, J.D. (1994). The toxicology of aflatoxins. Human health, veterinary and agricultural significance. pp. 6-8 Academic press, San Diego, Ca. Edds, G.T. & Bortell, R.A. (1983). Biological effects of aflatoxins-poultry. pp. 56-61. In:L U.L. Diener, R.L. Asquity and J.W. Dickens Eds. Aflatoxin and A. flavus in corn AAES, Auburn Univ, Alabama Edds, G.T.; Nair, N.P. & Simpson, C.F. (1973). Effect of AFB1 on resistance in poultry against cecal coccidiosis and Marek's disease. Am. J. Vet. Res, Vol.34, pp.819-826 FDA, Food and Drug Administration (1979). Conference on mycotoxins in animal feeds and grains related to animal health. Rockville, Maryland Fehr, P.M. & Delage, J. (1970). Effect de l'aflatoxine sur les fermentations du rumen. Can. Nutr. Diet, Vol.5, pp59-61 Fouzia Begum. & Samajpati.N(2000). Mycotoxins production on rice, pulses and oilseeds. Naturwissenschaften, Vol.87, pp.275-277 Garlich, J.D.; Tung, H.T. & Hamilton, P.B. (1973). The effects of short term feeding of aflatoxin on egg production and some plasma constituents of laying hens. Poult. Sci, Vol.52, pp.2206. Gewurz, H. & Suyehira, L.A. (1976). Manual of clinical immunology. Rose, N.R. and Friedman, H.R. Eds., American Society of Microbiologists, Washongton, D.C., 36. Giambrone, J.J.; Diener, U.I. Davis, N.D. Panangala, V.S. & Hoerr, F.J. (1985). Effects of aflatoxin on young turkeys and broiler chickens. Poult. Sci, Vol.64, pp.1678-1684 Giambrone, J.J.; Ewert, D.L. Wyatt, R.D. & Eidson, C.S. (1978a). Effect of aflatoxin on the humoral and cell-mediated immune systems of chicken. Am. J. Vet. Res,Vol.39, pp. 305 Giambrone, J.J.; Partadiredja, M. Eidson, C.S. Kleven, S.H. and Wyatt, R.D. (1978b). Interaction of aflatoxin with infectious bursal disease virus infection in young chickens. Avian Dis,Vol.22, pp.431

248

Aflatoxins – Biochemistry and Molecular Biology

Gillespie, J.R.& Tyler, W.S. (1969). Chronic alveolar emphysema in the horse. Advances in Veterinary Science and Comparative Medicine, Vol.13, pp.59–99 Glahn, R.P. (1993). Mycotoxins and the avian kidney: Assessment of physiological function.wld. Poult. Sci. J, Vol.49, pp.242-250 Greene, H.J.& Oehme, F.W. (1976). A possible case of equine aflatoxicosis. Clinical Toxicology, Vol.9, pp.251–254 Groopman, J.D.; Donahue, P.R. Zhu, J. Chen, J. & Wogan, G.N. (1985). Aflatoxin metabolism in humans. Detection of metabolites and nucleic acid adducts in urine by affinity chromatography. Proc. Natl. Acad. Sci., USA, Vol.82, pp.6492-6497 Guthrie, L.D. (1979). Effect of aflatoxin in corn on production and reproduction in dairy cattle. J. Dairy Sci, Vol.62, pp.134 Halliwell, R.E.W.; McGorum, B.C. Irving, P. & Dixon, P.M. (1993). Local and systemic antibody production in horses affected with chronic obstructive pulmonary disease. Veterinary Immunology and Immunopatholog, Vol.38, pp. 201–215 Hamilton, P. B.& Harris, J. R. (1971). Interaction of aflatoxicosis with Candida albicans: infections and other stresses in chickens. Poult. Sci, Vol. 50, pp.906-912 Hamilton, P.B. & Garlich, J.D. (1972). Failure of vitamin supplementation to alter the fatty liver syndrome by aflatoxin. Poult. Sci, Vol.51, pp.688 Hamilton, P.B. (1971). A natural and extremely severe occurrence of aflatoxicosis in laying hens. Poult. Sci, Vol.50, pp.1880-1882 Hartley, R. D.; Nesbitt, B. F. & O'Kelly, J. (1963). Toxic metabolites of Aspergillus flavus. Nature, Vol.198, pp.1056-1058 Hatch, R.C.; Clark, J.D. Jain, A.V. & Mahaffey, E.A. (1971). Experimentally induced acute aflatoxicosis in goats treated with ethyl maleate glutathione precursors or thiosulfate. Am. J. Vet. Res, Vol.40, pp.405-411 Hegazy, S.M.; Azzam, A. & Gabal, M.A. (1991). Interaction of naturally occurring aflatoxins in poultry feed and immunization against fowl cholera. Poult. Sci, Vol.70, pp. 24252428 Hesseltine, C.W. (1983). Introduction, definition and history of mycotoxins of importance to animal production. In: Interactions of mycotoxins in animal production. National Academy of Science, Washington, DC. Hetzel, D.J.S.; Hoffmann, D. Van De Ven, J. & Soeripto, S. (1984). Mortality rate and liver histopathology in four breeds of ducks following long term exposure to low levels of aflatoxins. Sing. Vet. J, Vol.8, pp6-14 Holzapfel, C. W.; Steyn, P. S. & Purchase, I. F. H. (1966). Isolation and structure of aflatoxins M I and M 2. Tetrahedron Lett,Vol. 25, pp.2799-2803 Horn, C.W.; Boleman, L.L. Coffman, C,G. Deton, J.H. & Lawhorn, D.B. (1989). Mycotoxins in feed and food producing crops college state Texas. Texas Vet. Med. Diagnostic, The National Dairy Database (1992) Horr, F.J. & D'Andrea, G.H. (1983). Biological effects of aflatoxin in swine. pp.51-55. In: U.L. Diener; R.L. Asquith and J.D. Dickens Eds. Aflatoxin and A. flavus in corn AAES, Aubura Univ. Alabama

Aflatoxins and Aflatoxicosis in Human and Animals

249

Howarth, B.Jr. & Wyatt, R.D. (1976). Effect of dietary aflatoxin on fertility, hatchability and progeny performance of broiler breeder hens. Appl. Environ. Microbiol, Vol.31, pp.680-684 Huff, W.E. & Doerr, J.A. (1980). Synergism between aflatoxin and OA in broiler chickens. Poult. Sci, Vol.60, pp.55 Huff, W.E.; Doerr, J.A. Wabeck, C.J. Chaloupka, O.W. May, J.D. & Merkley, J.W. (1983). Individual and combined effects of aflatoxin and ochratoxin A on bruising in broiler chickens. Poult. Sci, Vol.62, pp.1764-1771 Huff, W.E.; Kubena, R.B. Harvey, R.H. Corrier, D.E. & Mollenhaur, H.H. (1986). Progression of aflatoxicosis in broiler chickens. Poult. Sci, Vol.65, pp.1891-1899 Huff, W.E.; Wyatt, R.D. & Hamilton, P.B. (1975). Effects of dietary aflatoxin on certain egg yolk parameters. Poult. Sci, Vol.54, pp.2014-2018 Humphreys, D.J. (1988). Veterinary toxicology.3rd ed. pp. 286, Bailiere Trindell, London Hutjens, M. (1983). Aflatoxin contaminated feed and dairy. In: J. Jacobsen, Aflatoxin 83. Texas Vet. Med. Diagnostic lab., Texas collage. Jayabarathi, P. & Mohamudha Parveen, R. (2010). Biochemical and histopathological analysis of aflatoxicosis in Growing hens fed with commercial poultry feed. International Journal of Pharmaceutical Sciences Review and Research , Vol.3, No.2, pp. 127-130 Jones, F.T.; Beth, M. Genter, M.M. Hagler, W.M. Hansen, J.A. Mowrewy, B.A. Poore, M.H. & Whitlow, L.W. (1994). Understanding and coping with effects of mycotoxins in livestock feed and forage. Electronic publication No. DRO-29, NCCES, North Carolina State Univ., Raleigh, North Carolina. Jones, F.T.; Hagler, W.H. & Hamilton, P.B. (1982). Association of low levels of aflatoxin in feeds with productivity losses in commercial broiler operations. Poult. Sci, Vol.6, pp. 861-868 Jones, N.R. & Jones, B.D. (1978). Aflatoxins. Voeding, Vol.30, pp.330 Kato, R., Takaoka, A. Onoda, K. & Omori, Y. (1970). Different effect of aflatoxin on the induction of tryptophan oxygenase and of microsomal hydroxylase system. J. Biochem, (Tokyo). Vol.68, pp.589-592 Keeler, R.F. & Tu, A.T. (1983). Handbook of natural toxins. Vol. I. Plant and fungal toxin, pp.308, New York, Marcal Dekker, Ine Kiemeier, F. & Buchner, M. (1977). Zur verteilung van aflatoxin M1 auf Molke and Bruch bei der Kasehersteilung. Z. Lebensm. Unters. Forsch, Vol.164, pp.87 Krishnamachari, K.A.; Bhat, R.V. Nagarajan, V. & Tilak, T.B. (1975a). Investigations into outbreak of hepatitis in parts of Western India. Indian J. Med. Res, Vol.63, pp.10361049 Krishnamachari, K.A.; Nagarajan, V.; Bhat, R.V. & Tilak, T.B. (1975b). Hepatitis due to aflatoxicosis. An outbreak in Western India. Lencet i. pp.1061-1062 Lamont, M.H. (1979). Cases of suspected mycotoxicosis as reported by veterinary investigation centers. Proc. Mycotoxins Anim. Dis, Vol.3, pp.38-39 Lanza, G.M.; Washburn, K.W. & Wyatt, R.D. (1980). Strain variation in hematological response of broilers to dietary aflatoxin. Poult. Sci, Vol.59, pp.2686-2691

250

Aflatoxins – Biochemistry and Molecular Biology

Larsson, P., Persson, E., Tydén, E. & Tjälve, H. (2003). Cell-specific activation of aflatoxin B1 correlates with presence of some cytochrome P450 enzymes in olfactory and respiratory tissues in horse. Research in Veterinary Science, Vol.74, pp.227–233 Lawlor, P.G. & Lynch, P.B. (2001). Mycotoxins in pig feeds. 2: clinical aspects. Irish Vet. J, Vol.54, No.4, pp.172-176 Leeson, S.; Diaz, G.J. & Summers, J.D. (1995). Poultry metabolic disorders and mycotixns. pp. 249-298, University Books, Guelph, Ontario, Canada Liggett, A.D.; Colvin, B.M. Beaver, R.W. & Wilson, D.M. (1986). Canine aflatoxicosis: A continuing problem. Vet. Hum. Toxicol, Vol.28, pp.428-430 Lillehoj, E.B. (1983). Effect of environmental and cultural factors of aflatoxin contamination of developing corn kernels. In: V.L. Diener, R.L. Asquith and J.W. Dickens (Eds). Aflatoxin and A. flavus in corn. Southern Coop Serv. Bull. 279, Craftmaster, Opelika, Ala, 112P. Lynch, G.P. (1972). Mycotoxins in feedstuffs and their effect on dairy cattle. J. Dairy Sci, Vol.55, pp.1243-1255 Masri, M. S.; Booth, A. N. and Hsieh, D. P. H. (1974). Comparative metabolic conversion of aflas B1 to aflatoxin M1 and aflatoxin Q by monkey, rat, and chicken. Life Sci, Vol.15, pp.203-212 Masri, M.S.; Catcia, V.C. & Page, J.R. (1969). AFM content of milk from cows fed known amounts of aflatoxin. Vet. Rec, Vol.84, pp.146-147 Meerdink, G.L. (2002). Mycotoxins. Clinical Techniques in Equine Practice 1. pp.89–93 Mintzlaff, H. J., Lotzsch, R. Tauchmann, F. Meyer, W. & Leistner, L. (1974). Aflatoxin residues in the liver of broiler chicken given aflatoxin-containing feed. Fleischwirtschaft, Vol.54, pp.774-778 Mohiuddin, S.M.; Reddy, M.V. Reddy, M.M. & Ramakrishna, K. (1986). Studies on phagocytic activity and haematological changes in aflatoxicosis in poultry. Indian Vet. J, Vol.63, pp.442-445 Morchouse, L.G. (1981). Mycotoxins-their toxicology and principle lesions. Proc. U.S. Anim. Health Assoc. pp.232. Muller, R.D.; Carlson, C.W. Semeniuk, G. & Harshfield, G.S. (1970). The response of chicks, duckling, goslings, pheasants and poults to graded levels of aflatoxins. Poult. Sci, Vol.49, pp.1346-1350 Nabney, J.; Burbage, M. B. Allcroft, R. and Lewis, G. (1967). Metabolism of aflatoxin in sheep. Excretion pattern in the lactating ewe. Food Cosmet. Toxicol.Vol.5. pp.11-17 Nesbitt, B.; O’Kelly, K.; Sargeant, K. & Sheridan, A. (1962). Toxic metabolites of Aspergillus flavus. Nature, Vol.195, pp.1062-1063 Newberne, P.M.; Russo, R. and Wogan, G.N. (1966). Acute toxicity of AFB1 in the dog. Pathol. Vet. Vol.3. pp.331-340. Nibbelink, S.K. (1986). Aflatoxicosis in food animals. A clinical review. Iowa State Univ.Vet. Vol.48, pp. 28-31. Nesheim, M.C. & Ivy, C.A. (1971). Effect of aflatoxin on egg production and liver fat in laying hens. Cornell Nutr. Conf. Buffalo, pp. 126-129. Okoye, J.O.A.; Asuzu, I.U. & Gugnani, J.C. (1988). Paralysis and lameness associated with aflatoxicosis in broilers. Avian Pathol, Vol.17, pp.731-734

Aflatoxins and Aflatoxicosis in Human and Animals

251

Osborne, D.J.; Huff, W.E. Hamilton, P.B. & Burtmeister, H.R. (1982). Comparison of ochratoxin, aflatoxin and T-2 toxin for their effects on selected parameters related to digestion and evidence for specific metabolism of carotenoids in chickens. Poult. Sci, Vol.61, pp.1646-1652. Osweiller, G.D. (1992). Mycotoxins. In: Diseases of swine.7th ed. pp.735-743. Edited by A.D. Leman, B.E. Straw, W.L. Mengeling, S.D.; Allaire & D.J. Taylor Wolfe publishing, London Patterson, D.S.P. & Roberts, B.A. (1970). The formation of aflatoxin B2a and G2a and their degradation products during the in vitro detoxification of aflatoxin by livers of certain avian and mammalian species. Food cosmet. Toxicol, Vol. 8, pp. 527-538. Patterson, D.S.P. & Allcroft, R. (1970). Metabolism of aflatoxins in susceptible and resistant animal species. Fd. Cosmet. Toxicol, Vol.8, pp.43 Patterson, D.S.; Galaney, E.M. & Roberts, B.A.(1978). The estimation of AFM1 in milk using 2-dimensional TLC. Fd.Cosmet toxicol, Vol.16, pp.49-50 Pier, A.C. (1973). Effects of aflatoxin on immunity. J. Amer. Vet. Med. Assoc, Vol.163, pp.12681269 Pier, A.C.; Cysewski, S.J. Richard, J.L. Baetz, A.L. & Mitchell, L. (1976). Proc. U.S., Anim. Hlth. Assoc, Vol.80, pp.130. In: Mackenzie et al. (1981) Price, R.L.; Paulson, J.H. Lough, O.E. Gingg, C. & Kurtz, A.G. (1985). Aflatoxin conversion by dairy cattle consuming naturally contaminated whole cottonseed. J. Fd. Prot, Vol.48, pp.11-15 Radostits, O.M.; Gay, C.C. Blood, D.C. & Hinchcliff, K.W. (2000). Veterinary medicine, pp.1684-1688, W.B. Saunders Co. Ltd., London. Raisbeck, M.F.; Rottinghaus, G.E. & Kendall, J.D. (1991). Effects of naturally occurring mycotoxins on ruminants. pp. 647-677. Rao, V.N. & Joshi, H.C. (1993). Effect of certain drugs on acute induced aflatoxicosis in chicken (4 mg AFB1/ kg b.wt.). Ind. Vet. J, Vol 70, pp.344-347 Ray, A.C.; Abbitt, B. Cotter, S.R. Murphy, M.J.; Reagor, J.C. Robinson, R.M. West, J.E. and Whitford, H.W. (1986). Bovine abortion and death associated with consumption of aflatoxin contaminated peanuts. J. Am. Vet. Med. Assoc, Vol.184, pp.956 Reagor, J.C. (1996). Implications of mycotoxins in horses. WEVR, 96, Cybersteed, Ine. Reddy, D.N.; Rao, P.V. Reddy, V.R. and Yadgiri, B. (1984). Effect of selected levels of dietary aflatoxin on the performance of broiler chickens. Indian, J. Anim. Sci,Vol.54, pp.6873 Richard, J.L.; Pier, A.C. Stubblefiels, R.D. Shotwell, O.L. Lyon, R.L. and Cutlip, R.C. (1983). Effect of feeding cornnaturally contaminated with aflatoxin on feed efficiency, on physiology, immunologic and pathologic changes and in tissue residues in steers. Am. J. Vet. Re, Vol.44, pp.1294 Richard, L.J.; Stubblefield, R.D. Lyon, R.L. Peden, W.M. Thurston, J.R. & Rimler, R.B. (1987). Distribution and clearance of aflatoxin B1 and M1 in turkeys fed diets containing 50 or 150 ppb aflatoxin from naturally contaminated corn. Avian Dis, Vol.30, pp.788793

252

Aflatoxins – Biochemistry and Molecular Biology

Richard, T.L.; Thurston, J.R. & Pier, A.C. (1975). Mycotoxin-induced alterations in immunity. In: Microbiology, Schlessinger, D. Ed., American Society of Microbiologist, Washington, D.C. Richardson, K.E. & Hamilton, P.B. (1987). Enhanced production of pancreatic digestive enzymes during aflatoxicosis in egg-type chickens. Poult. Sci, Vol.66, pp.1470-1478 Rodricks, J.V. & Stoloff, L. (1977). Mycotoxins in human and animal health. Pathotox publishers, park forest, south, 1L, pp.67-69 Royes, J.B. & Yanong, R.P. (2002). Molds in fish feeds and aflatoxicosis. Copyright by the University of Florida, Institute of Agric. Sci. (UF/ IFAS) Rumbeiha, W. (2001). Mycotoxicosis in pets. DCPAH-Newsletter.Vol.18, No. 2. Samarajeewa, U.; Arseculeratne, S.N. & Tennekoon, G.E. (1975). Spontaneous and experimental aflatoxicosis in goats. Res. Vet. Sci, Vol.19, pp.269-277 Sargeant, K.; Carraghan, R.B. & Allcroft, R. (1963).Toxic products in groundnuts. Chemistry and origin. Chem. And Ind, pp.53-55 Sargeant, K.; Sheridan, J.; O’Kelly, J. & Carnaghan, R. B. A. (1961). Toxicity associated with certain samples of groundnuts. Nature, Vol.192, pp.1096-1097 Sawhney, D.S.; Vadehra, D.V. & Baker, R.C. (1973a). Aflatoxicosis in the laying Japanese quail. Poult. Sci, Vol.52, pp.485-493 Sawhney, D.S.; Vadehra, D.V. & Baker, R.C. (1973b). The metabolism of 14C aflatoxins in laying hens. Poult. Sci, Vol.52, pp.1302-1309 Schabort, J. C. & Steyn, M. (1969). Substrate and Phenobarbital inducible aflatoxin-4hydroxylation and aflatoxin metabolism by rat liver microsomes. Biochem. Pharmacol, Vol.1, pp.2241-2252 Shank, R. C. & Wogan, G. N. (1966). Acute effects of aflatoxin B1 on liver composition and metabolism in the rat and duckling. Toxicol. Appl. Pharmacol, Vol.9, pp.468-476. Shank, R.C. (1977). Environmental cancer. pp.291-318. In: H.F. Kraybill and M.A. Mehlman (eds.), John Wiley sons Ltd New York. Shankaran, R.; Raj H. G. & Venkatasubramanian T. A. (1970). Effect of aflatoxin on carbohydrate metabolism in chick liver. Enzymlogia, Vol.39, pp.371-378 Sharlin, J.S.; Howarth, B.Jr. Thompson, E.N. and Wyatt, R.D. (1981). Decreased reproductive potential and reduced feed consumption in nature white leghorn males fed aflatoxin. Poult. Sci, Vol.60, pp.2701 Sharma, R.P. (1993). Immunotoxicity of mycotoxins. J. Dairy Sci, Vol.76, pp.892-897 Siller, W.G. & Ostler, D.C. (1961). The histopathology of an entero-hepatic syndrome of turkey poults. Vet. Rec, Vol.73, pp.134-138 Sisk, D.B.; Carlton, W.W. and Curtin, T.M. (1968). Experimental aflatoxicosis in young swine. Am. J. Vet. Res, Vol.29, pp.207-215 Slowik, J.; Graczyk, S. and Madej, J.A. (1985). The effect of single dose of AFB1 on the value of nuclear index of blood lymphocytes and on histopathological changes in the liver, bursa of fabricious, suprarenal glands and spleen in ducklings. Folia Histochem. Cytobiol Vol.3. pp.71-80. Smith, B.P. (2002). Large animal internal medicine. 3rd ed. pp.1627-1637. Mosby, Inc. USA. Smith, J. W.; Prince, W. R. & Hamilton, P. B. (1969). Relationship of aflatoxicosis to Salmonella gallirarem infections of chickens. Appl. Micobiol, Vol.18, pp.946-947

Aflatoxins and Aflatoxicosis in Human and Animals

253

Smith, J.E. & Hamilton, P.B. (1970). Aflatoxicosis in the broiler chicken. Poult. Sci, Vol.49, pp.207 Smith, R. H. (1965). The inhibition of amino acid activation in liver and E. coli preparations by aflatoxin in vivo. Biochem. J, Vol.95, pp.438-448 Sorensson, W.G.; Simpson, J.P. Peach, M.J. Thedell, T.D. & Olenchock, S.A. (1981). Aflatoxin in respirable dust particles. Journal of Toxicology and Environmental Health, Vol.7, pp.669–672 Stewart, D. & Larson, E. (2002). Aflatoxicosis in wildlife. Information Sheet 1582 Mississippi State Univ. Extension Service., Cooperating with U.S. Dept. of Agriculture. Stewart, R.G.; Skeeles, J.K. Wyatt, R.D. Brown, J. Page, R.K. Russell, I.D. & Lukert, P.D. (1985). The effect of aflatoxin on complement activity in broiler chickens. Poult. Sci, Vol.64, pp.616-619 Stoloff, L. & Trucksess, M.W. (1979). Distribution of aflatoxins B1 and M1 in contaminated calf and pig livers. Journal of the Association of Official Analytical Chemists, Vol.62, pp1361–1362 Stoloff, L. (1980). Aflatoxin M1 in perspective. J. Food Prot, Vol.43, pp.226 Thaxton, J.P.; Tung, H.T. & Hamilton, P.B. (1974). Immunosuppression in chickens by aflatoxin. Poult. Sci, Vol.53, pp.721 Tung, H.T.; Cook, F.W. Wyatt, R.D. & Hamilton, P.B. (1975a). The anemia caused by aflatoxin. Poult. Sci, Vol.54, pp.1962-1969 Tung, H.T.; Wyatt, R.D. Thaxton, P. & Hamilton, P.B. (1973). Impairment of kidney function during aflatoxicosis. Poult. Sci, Vol.52, pp.873 Tung, H.T.; Wyatt, R.D. Thaxon, P. and Hamilton, P.B. (1975b). Concentrations of serum proteins during aflatoxicosis. Toxicol. Appl. Pharmacol, Vol.34, pp.320-326 Tydèn, E.; Olsèn, L. Tallkvist, J. Tjälve, H.& Larsson, P. (2008). Cytochrome P450 3A, NADPH cytochrome P450 reductase and cytochrome b in the upper airways in horse. Research in Veterinary Science, Vol.85, pp.80–85 Van der Linde, L.A.; Van der Frens, A.M. & Van Esch, G.J. (1965). Experiments with cows fed groundnut meal containing aflatoxin. pp. 247. In: G.N. Wogan, ed. Mycotoxins in foodstuffs. MIT Press, Cambridge, mass. Van Der Zijden, A. S. M.; Koelensmid, W. A. A. Boldingh, J. Barrett, C. B. Ord, W. 0. W. 0. & Philip, J. (1962). Isolation in crystalline form of a toxin responsible for turkey X disease. Nature, Vol.195, pp.1060-1062 Van dorp, D. A.; Van Der Zijden, A. S. M. Beerthuis, R. K. Sparreboom, S. Ord, K. De Jong, & Keuning, R. (1963). Dihydroaflatoxin B, a metabolite of Aspergillus flavus Remarks on the structure of aflatoxin B. Rec. Trav. Chim, Vol.82, pp.587-592 Van dorp, D. A.; Van der zijden, A. S. M.; Beerthuis, R. K.; Sparreboom, S.; Ord, W. 0.; De jong, K.& Keuning, R..(1963) Dihydroaflatoxin B, a metabolite of Aspergillus flavus. Remarks on the structure of aflatoxin B. Rec. Trav. Chim. Vol. 82, pp.587-592 Veltmann, J. R. (1984). Reducing effects of mycotoxins through nutrition. Poult. Digest, pp. 190-194. Vesonder, R.; Haliburton, J. Stubblefield, R. Gilmore, W. & Peterson, S. (1991). Aspergillus flavus and aflatoxin B1, B2 and M1 in corn associated with equine death. Archives of Environmental Contamination and Toxicology, Vol.20, pp.151–153

254

Aflatoxins – Biochemistry and Molecular Biology

Voight, M. N.; Wyatt, R.D.; Ayers, J.C. and Koehler, P. (1980). Abnormal concentrations of B vitamin and amino acids in plasma, bile and liver of chicks with aflatoxicosis. Appl. Environ. Microbiol, Vol. 40, pp. 870-875 Wannop, C.C. (1961). The histopathology of turkey "x" disease in great Britain. Avian Dis, Vol.5, pp. 371-381 Weidenborner, M. (2001). Encyclopedia of food mycotoxins."Spronger Publisher Berlin, New York, London. WHO, World Health Organization (1979). Environmental Health Criteria, Safety evaluation of certain food additives. pp. 1-127 Wilson, H.R.; Douglas, C.R. Harms, R.H. & Edds, G.T. (1975). Reduction of aflatoxin effects on quail. Poult. Sci, Vol.54, pp.923-925 Wogan, N. (1966). Chemical nature and biological effects of the Aflatoxins. Bacteriological Review, Vol.30, No.2, pp.460-470 Wyatt, R.D. & Hamilton, P.B. (1975). Interaction between aflatoxicosis and a natural infection of chickens with Salmonella. Appl. Microbiol, Vol.30, pp.870-872 Wyatt, R.D. (1991). Poultry, pp.553-578, In: Smith, J.E. and Henderson, R.S. (1991). Wylie, T.D. & Morehouse, L.G. (1978). Mycotoxic fungi, mycotoxins and mycotoxicosis. Vol.2, pp.414-427. Marcel Dekker, INC, USA

13 Aflatoxins and Their Impact on Human and Animal Health: An Emerging Problem Ernesto

Moreno-Martínez2

Eva G. Lizárraga-Paulín1, and Susana P. Miranda-Castro1

1Laboratorio

de Biotecnología, Facultad de Estudios Superiores Cuautitlán, UNAM. Cuautitlán Izcalli, Estado de México, 2Unidad de Investigación en Granos y Semillas, Facultad de Estudios Superiores Cuautitlán, CAT, UNAM. Cuautitlán Izcalli, Estado de México, México 1. Introduction Aspergillus is a fungus that essentially belongs to grains storage flora. It grows optimally at 25 °C with a minimum necessary water activity of 0.75. It starts to produce secondary metabolites at 10-12 ºC, but the most toxic ones are produced at 25°C with a water activity of 0.95 (Hesseltine 1976). Those toxic secondary metabolites named aflatoxins (AF) is a group of mycotoxins produced by a large number of Aspergillus species, basically by three phylogenetically distinct sections. The main producers are A. flavus, and A. parasiticus, but it has been demonstrated that A. nomius, A. pseudotamarii, A. parvisclerotigenus, and A.bombycis of section Flavi, A. ochraceoroseus and A. rambellii from section Ochraceorosei and Emericella astellata and E. venezuelensis from Nidulatans section also generate aflatoxins (IARC 2002; Frisvad et. al., 2005). All of them contaminate a large fraction of the world’s food, including maize, rice, sorghum, barley, rye, wheat, peanut, groundnut, soya, cottonseed, and other derivative products made from these primary feedstuffs in low-income countries (Rizzi et al., 2003; Saleemullah et al., 2006; Strosnider et. al., 2006; Masoero et. al., 2007; Caloni, 2010). Although aflatoxins have been a problem throughout history, until 1960 they have been recognized as significant contaminants within agriculture, because in this year they were initially isolated and identified as the causative toxins in “Turkey-X-disease” after 100,000 turkeys died in England from an acute necrosis of the liver and hyperplasia of the bile duct after consuming groundnuts infected with Aspergillus flavus (Asao et. al., 1965; D’Mello, 1997; Strosnider et. al., 2006). Williams et al. estimated in 2004 that 4.5 billion of the world’s population is exposed to aflatoxins because they are also everywhere. Some essential factors that affect aflatoxin contamination include the climate of the region, the genotype of the crop planted, the soil type, the minimum and maximum daily temperatures, and the daily net evaporation (Strosnider et. al., 2006). Moreover, aflatoxin contamination is also promoted by stress or damage to the crop due to drought before harvest, the insect activity, a poor timing of harvest, the heavy rains during and after harvest, and an inadequate drying of the crop before storage. Levels of humidity, temperature, and aeration during storage are also important factors that are

256

Aflatoxins – Biochemistry and Molecular Biology

intimately related with the actual problems of climate changes and environmental warming around the whole world (Cotty & Jaime-García, 2007; Paterson & Lima, 2010). There have been identified 18 types of aflatoxins, nevertheless, the naturally occurring and well-known ones are aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1) and aflatoxin G2 (AFG2) (Gimeno, 2004; Saleemullah et. al., 2006; Strosnider et. al., 2006). These names were given due to their blue (B) or green (G) fluorescence properties under ultraviolet light and their migration patterns during chromatography (Wogan & Busby, 1980; Dikeman & Green, 1992). The International Agency for Research on Cancer (IARC, 2000) has classified aflatoxin B1 as a group 1 carcinogen (that means carcinogenic to humans) since 1987, and a group 1 carcinogenic agent since 1993 due to the exposure to hepatitis B virus (Castegnaro & McGregor, 1998). AFB1 is the most prevalent aflatoxin usually found in cases of aflatoxicosis, and is responsible for acute toxicity, chronic toxicity, carcinogenicity, teratogenicity, genotoxicity and immunotoxicity. AFM1 is a metabolic derivate of AFB1, and AFM2 is a metabolic derivate of AFB2; both come from the metabolism of some animals, and are normally found in milk and urine (Gimeno, 2004; Strosnider et. al., 2006). The B-toxins are characterized by the fusion of a cyclopentenone ring to the lactone ring of the coumarin structure, while G- toxins contain an additional fused lactone ring. Aflatoxin B1 and to a lesser extent AFG1, are responsible for the biological potency of aflatoxincontaminated feed. These two toxins possess an unsaturated bond at the 8,9 position on the terminal furan ring. Aflatoxin B2 and AFG2 are essentially biologically inactive unless these toxins are first metabolically oxidized to AFB1 and AFG1 in vivo (Verma, 2004). AFM1 and AFM2 are hydroxylated derivatives of AFB1 and AFB2 that may be found in milk, milk products or meat (hence the designation M1). They are formed by the metabolism of B1 and B2 in the body of the animals following absorption of contaminated feeds (Gimeno, 2004; Verma, 2004; Wild & Gong, 2010). In animals, aflatoxins impair growth and are immunosuppressive. B aflatoxin has been reported to induce liver and kidney tumors in rodents, and there has been found a possible link to increased esophageal cancer. Aflatoxins have been recently considered as an important sanitary problem because it has been demonstrated that human exposure to mycotoxins may result from consumption of plant derived foods that are contaminated with toxins and their metabolites (which are present in animal products such as milk, meat, visceral organs and eggs) or exposure to air and dust containing toxins (Jarvis, 2002). It has been reported that aflatoxins, once ingested (because of their low molecular weight), are rapidly adsorbed in the gastro-intestinal tract through a non-described passive mechanism, and then quickly appear as metabolites in blood after just 15 minutes and in milk as soon as 12 hours post-feeding (Yiannikouris & Jouany, 2002; Moschini et. al., 2006). Aflatoxins are hepatocarcinogenic particularly in conjunction with chronic hepatitis B virus infection, and cause aflatoxicosis in episodic poisoning outbreaks. Recent studies also suggest that the B aflatoxins may cause neural tube defects in populations that consume maize as a staple food (Wild & Gong, 2010). Due to this important global issue, some organizations and institutions have been purposing a great number of practical primary and secondary prevention strategies, especially for developing countries, in order to reduce the risks given by this public problem, but they could be beneficial if political wills and financial investments are applied to what remains a largely ignored worldwide health matter.

Aflatoxins and Their Impact on Human and Animal Health: An Emerging Problem

257

2. Aflatoxins in food products from contaminated grains In many low-income countries, mycotoxins, and particularly aflatoxins, affect staple foods including cereals (maize, wheat and rice principally) and their derivates; oilseeds (cotton, peanut, rapeseed, coconut, sunflowers and others), cassava, groundnuts and other nuts, and a great variety of foods which are consumed by humans like dry fruits, delicatessen products, spices, wines, legumes, fruits, milk and milk derivates (Gimeno, 2004; Wild & Gong 2010). Maize and groundnuts are major sources of human exposure because of their greater susceptibility to contamination and frequent consumption throughout the world. Table 1 shows some of the most important commodities affected by aflatoxins producer species, according to a review made by Abdin and collaborators in 2010. Type of aflatoxin

Producer fungal species

Affected commodities

B (B1, B2)

A. flavus, A. parasiticus, A. tamarii, A. pseudotamarii, A. bombycis, A. parvisclerotigenus, A. nomius, A. minisclerotigenes, A. oryzae, A. toxicarius, A. versicolor, A. rambellii, A. arachidicola, A. ochraceoroseus, Emericella astellata, E. venezuelensis.

Cotton seed, peanuts, peanut butter, pea, sorghum, rice, pistachio, maize, oilseed rape, maize flour, sunflower seed, figs, spices, meats, dairy products, fruit juices (apple, guava)

G (G1, G2)

Peanuts, cotton seed, sunflower seed, A. parasiticus, A. nomius, A. tree nuts, pistachio, peanut butter, bombycis, A. pseudotamarii, A. maize flour, pea, cereals, corn, figs, terreus, A. versicolor, A. arachidicola, meats, spices, dairy products, fruit A. toxicarius, A. minisclerotigenes. juices (apple, guava)

Table 1. Major commodities affected by aflatoxins (Taken from Abdin et. al., 2010.) Aflatoxins are most prevalent in latitudes between 40° N and 40° S of the equator, but the greatest health risk lies within developing countries in tropical regions, which rely on these commodities as their staple food source (Strosnider et. al., 2006). Even, in some processed typical food like Mexican pozol, there have been found large amounts of aflatoxins, being the AFB2 the more prevalent and abundant toxin, suggesting that AFB2 is more resistant than AFB1 to the alkaline conditions given during hard processes like nixtamalization (Kamimura, 1989; Méndez & Moreno, 2004). In wealthy grain-producing countries of the world, economic resources exist to ensure that regulations to limit aflatoxin exposure in the food supply are implemented. Furthermore, in markets of grain commodities, the prices of corn and groundnuts are often dictated by aflatoxin content, which contributes to lower levels of exposure in wealthy countries. Thus, a result of these regulations and market forces is that people in economically developing countries are exposed to far higher levels of aflatoxins in the diet (Groopman et. al., 2008). The presence of aflatoxins in food means a risk for both animals and human beings. This is because not only grains (generally consumed by people), but also whole plants and grasses from which they emerge, could be contaminated by mycotoxins. This is a serious threat for animals, particularly livestock, because the herbaceous food they consume (commonly known as ensilage or forage) contains a large amount of aflatoxins, particularly if field was contaminated. A potentially hazardous feed is ground high-moisture corn, unless it is

258

Aflatoxins – Biochemistry and Molecular Biology

treated with adequate preservatives (e.g., propionates); the moisture content promotes the growth of the toxigenic molds and grinding of the kernel destroys the natural barrier to infestation. Hay (unless it contains a large complement of cereal grain infested in the field) is rarely if ever a source of appreciable aflatoxin (however, hay and forage may be sources of other mycotoxins such as ergot alkaloids, sporodesmin, slaframine, etc.). The fungus must gain access to susceptible parts of the plant (e.g., the corn kernel, cotton seed, etc.) before it grows and elaborates aflatoxins. Seasonal peaks in aflatoxin content are seen in key years when drought-damaged plants or insect-damaged crops are rendered more susceptible to fungal invasion. Wet harvest seasons also may contribute to high levels of aflatoxin in certain crops. Aflatoxin sometimes develops in crops stored at levels of moisture content > 15% or properly dried crops stored in leaky bins (Pier, 1992). Grains for animal feed in the United States are allowed 300 ppb aflatoxin, because this concentration not only provides protection against acute aflatoxicosis but also is low enough to allow most of the grain produced to be traded. In these animal feeding situations, the long-term risk of cancer is not a concern, except for the most susceptible species. Consequently, veterinary research has examined higher levels of exposure but for shorter time periods. This research provides most of the information on the toxicities of aflatoxin at intermediate rates of exposure (100–500 ppb) and is the most potentially relevant information that is appropriate for the human situation in developing countries where no control of aflatoxin is exercised. However, the differences between species in response to aflatoxin introduce a measure of speculation into the extension of farm animal–derived information to the human situation (Williams et. al., 2004).

3. Aflatoxins in food products from contaminated animals Aflatoxins M1 and M2 (whose names are derived from milk aflatoxins, and then related to meat aflatoxins too), are thermo-resistant hydroxylated metabolites produced by lactating animals consuming aflatoxin contaminated feeds. The ingested AFB1 and AFB2 are metabolized by livestock into AFM1 and AFM2 respectively, with estimated conversion ratio of 1–3% between AFB1 and AFM1 (Barbieri et. al., 1994; Ali et. al., 1999; Herzallah, 2009). The accepted limits of AFB1 and total aflatoxins in foods are 5 and 10 µg/kg, respectively, in more than 75 countries around the world whilst they are 2 and 4 µg/kg in the European Union (López et. al., 2003; Van Egmond & Jonker, 2004). The most alarming problem through time has been the presence of aflatoxin contaminated milk, because cows and goats (the major producers of drinking milk) are largely affected when eating contaminated forage all around the world (Helferich et. al., 1986; López et. al., 2003). By the way, it is important to consider that AFM1 concentrations in milk vary not only in the cow breed, but also in the concentration of AFB1 in the diet, the amount and duration of consumption of contaminated food and the animal health. There have been found differences between the amounts of AFM´s produced by different bovine species. In a review, Gimeno (2004) reports that in dairy cows, the relationship between the concentration of AFB1 in the final consumed ration and AFM1 excreted in breast milk could be 300:1; nevertheless this relation is only an approximation because the range is from 34:1 to 1600:1. In Holstein dairy cows consuming final rations with 80, 86, 470, 557, 1493 and 1089 µg of AFB1/Kg (ppb) on dry substance, there were found in milk AFM1 concentrations of 1.5, 0.245, 13.7, 4.7, 12.4 and 20.2 mg/L (ppb) respectively. On the other hand, when diet of Brindle cows was contaminated with 540 ppb of AFB1, 0.92 ppb of AFM1 was produced. In

Aflatoxins and Their Impact on Human and Animal Health: An Emerging Problem

259

other cows, the values of contamination in the diet ranged between 64 and 1799 ppb of AFB1 giving some residues in milk between 0.35 and 14.2 ppb of AFM1. With an intake of AFB1 for 2-60 mg / cow / day, AFM1 residues in milk could range between 1 and 50 pp. It is known that cows can transform AFB1 into AFM1 within 12-24 hours after ingestion of contaminated food. Even at six hours after ingestion, AFM1 residues can appear in milk, and the highest levels are reached after a few days. When the intake of AFB1 is stopped, the AFM1 concentration in the milk decreases to an undetectable level after 72 hours (Gimeno, 2004; Özdemir, 2007). Many studies have dealt with the transfer of AFB1 in milk as AFM1 when lactating animals ingested contaminated feed continuously, especially in cows. It has been suggested that an increase in AFM1 occurs due to Staphylococcus aureus infection and other bacterial infections related with somatic cells diseases (Veldman et. al., 1992; Masoero et. al., 2007). In contrast, little research has been conducted on the transfer of AFM1 into milk as a result of a single assumption of AFB1. From a practical standpoint, the use of highly contaminated feed by dairy farmers is unlikely; however, a single accidental feeding of contaminated feed may happen and can lead to milk AFM1 content above tolerance levels (Mazzete et. al., 2009). As mentioned before, goats are a clue target of aflatoxins too, so they have been studied as a good model for understanding the generating toxins metabolism (Smith et. al., 1994; Mazzete et. al., 2009). Mazzette and collaborators found that AFB1 ingested by lactating goats is quickly transferred to milk as AFM1. The maximum concentration of AFM1 was reached at 3-6 hours after the single oral administration of pure AFB1. Nevertheless, it showed a negative exponential trend and the toxin was no longer detected after 72 hours from administration. Therefore, an occasional oral assumption of AFB1 can lead to a transient contamination of AFM1 in goat’s milk. Milk has derivates that are consumed principally by humans. Among them we can find cheeses, butter, yogurt, cream and butterfat. The AFM1 distribution in some dairy foods made from contaminated milk is approximately: 40-60% in cheese, 10% in butterfat and 95% of AF production by A. flavus NRRL3357 without any obvious effect on fungal growth. Microarray-based gene expression profiling showed that expression of all genes in the AF biosynthetic cluster except for norB and the AF pathway regulatory gene, aflJ, were down-

Aflatoxins: Mechanisms of Inhibition by Antagonistic Plants and Microorganisms

291

regulated in caffeic acid-treated A. flavus compared with non-treated controls. Further microarray analysis of a number of genes involved in lipid metabolism, cell wall integrity and transport, and oxidative/antioxidative activities suggested a combination of events for the caffeic acid-induced inhibition of AF production by A. flavus (Kim et al., 2008). 3.5 Plant-based naturally occurring phenolics Phenolics are the most abundant secondary metabolites of plants with more than 8,000 known structures ranging from simple compounds such as phenolic acids to complex structures such as tannins (Dai & Mumper, 2010). Phenolic acids, flavonoids, tannins, stilbenes and lignans are the main classes of plant phenolics. Besides the role in defense against UV, pathogens, parasites and predators, plant polyphenols have received special attention regarding their potent antioxidant properties which make them promising in suppressing oxidative stress associated disorders such as cancer. Inhibition of fungal growth and AF production by phenolics has been a subject of many studies (Hua et al., 1999; Kim et al., 2005, 2006; RazzaghiAbyaneh et al., 2008). Hua et al. (1999) showed that plant phenolics i.e. acetosyringone, syringaldehyde and sinapinic acid efficiently inhibited the biosynthesis of AFB1 in A. flavus. Using a norsolorinic acid (NOR) accumulating mutant of A. flavus, they proposed that these phenolics exert their inhibitory effects on AFB1 biosynthesis at one or more early steps in the AF biosynthetic pathway. Razzaghi-Abyaneh et al. (2008) showed a novel biological activity from leaf essential oil of Satureja hortensis L. as strong inhibition of AF biosynthesis by A. parasiticus NRRL2999. The active substances purified by column chromatography were identified as phenolics, thymol and carvacrol. Bioassay with HPLC purified fractions revealed that both carvacrol and thymol effectively inhibited fungal growth. Inhibition of AFB1 and AFG1 production by these phenolics exhibited a dose-dependent manner at concentrations of 0.041 to 1.32 mM throughout all two-fold dilutions. The IC50 values of the compounds for AFB1 and AFG1 was as 0.50 and 0.06 mM. Since these phenolics are potent antioxidants, they likely exert their inhibitory activities on AF production through mediation of oxidative stress levels in the fungus. Kim et al. (2006), using a S. cerevisiae model system, demonstrated an effective synergism of natural phenolics with known antifungal chemicals such as carboxin and strobilurin. They showed that growth inhibition of A. flavus by the phenolics salicylic acid, thymol, vanillyl acetone, vanillin and cinnamic acid is via targeting the mitochondrial oxidative stress defense system. Since mitochondria are responsible for providing acetyl-CoA which is a main precursor for AF biosynthesis, disruption of mitochondrial respiration chain may account in part for the inhibitory effects of antifungal phenolics on AF production. 3.6 Azadirachta indica: A global tree for global problems Azadirachta indica A. Juss (syn. Melia azadirachta L., Neem, Margosa) is a subtropical tree native to the drier areas of Asia and Africa. The plant is known for its medicinal, spermicidal, antiviral, antibacterial, antiprotozoal, insecticidal, insect repellent, antifungal and antinematode properties (Allameh et al., 2001; Bhatnagar et al., 1988). It is indigenous to the Indian subcontinent where it has been used in agriculture, medicine and cosmetics. Several active substances from different parts of the plant have been identified. Extracts from different parts contain terpenoids, desactylimbin, quercetin and sitosterol. It has been shown that aqueous extracts of leaves and seeds inhibit AF production by A. parasiticus at concentrations higher than 10% (v/v) without affecting fungal growth. Studies suggested that the inhibitory components of these extracts are non-volatile substances that affect the

292

Aflatoxins – Biochemistry and Molecular Biology

synthesis of enzymes in the early steps of AF biosynthetic pathway (Bhatnagar et al., 1988; Zeringue and Bhatnagar, 1990). Allameh et al. (2001) did not find a positive correlation between AF production and the activity of fatty acid synthase, a key enzyme involved in AF production on neem-treated A. parasiticus. Razzaghi-Abyaneh et al. (2005) showed that AF production at 96 h in cultures containing 50% neem leaf and seed extracts was inhibited by 90 and 65%, respectively. Electron microscopy examination of treated fungus and nontreated controls revealed an association between decreased AF production and morphological changes suggesting that the integrity of cell barriers particularly cell wall is crucial in the regulation of AF biosynthesis and excretion. 3.7 Caffeine: An alkaloid from cocoa and coffee beans Caffeine is a xanthine alkaloid which was isolated from coffee in 1820 by a German chemist, Friedlieb Ferdinand Runge. This compound also is found in different quantities in the beans, leaves and fruits of some plants, and acts as a natural pesticide against plant pathogens. Caffeine has been reported to inhibit fungal growth and mycotoxin (sterigmatocystin, citrinin, patulin and ochratoxin A) production by some Aspergillus and Penicillium species (Buchanan & Lewis, 1984 and references therein). Its mechanism of action was elucidated by Buchanan & Lewis (1984). They observed nearly complete inhibition of AF production along with a marked suppression (80-90%) in growth of A. parasiticus in submerged cultures containing 2 mg/ml caffeine. Based on the results of the feeding experiments with [U-C14] glucose and enzymatic assays, Buchanan & Levis proposed that caffeine blocks AF production by affecting respiratory system of fungal cells and by inhibiting glucose uptake which is necessary for the production of acetyl-CoA, the building block of AFs. It seems that caffeine inhibits glucose uptake by directly affecting glucose transport system rather than altering the level or activity of enzymes associated with the glucose metabolism. 3.8 Gallic acid from walnuts Gallic acid is a phenolic compound and a key component of hydrolysable tannins found in different plant species such as walnuts, oat bark and tea leaves. It is synthesized from an early intermediate named 5-dehydroshikimate in shikimate pathway. Among diverse biological activities reported for gallic acid, antimicrobial, antioxidant and antitumor properties are involved in plant defense against environmental stressors and pathogens. Inhibition of AF prduction by gallic acid without obvious effect on fungal growth was first described by Cary et al. (2003). Investigation on the mechanisms of action of gallic acid has shown that the compound affects AF biosynthesis by i) inhibition of the expression of AF biosynthetic pathway genes nor1 and ver1 without affecting transcription of the regulatory gene i.e. aflR, ii) disruption of the signal transduction pathway of oxidative stress system and iii) suppression of expression of regulatory genes of AF biosynthetic pathway such as laeA, whose expression is triggered by oxidative stress (Cary et al., 2003; Kim et al., 2005; Mahoney & Molyneux, 2004). 3.9 Salicylaldehyde: A volatile natural plant compound Salicylaldehyde is an aroma compound of Fagopyrum esculentum and other buckwheat which acts as a key precursor of a variety of chelating agents with commercial importance. Little has been documented about physiological roles and biological properties of this volatile compound. Recently, Kim et al. (2010) showed that salicylaldehyde inhibits AF

Aflatoxins: Mechanisms of Inhibition by Antagonistic Plants and Microorganisms

293

production in A. flavus and A. parasiticus by 13-45% at a concentration of 9.5 mM; it also caused retardation in fungal growth. Using the model of yeast gene deletion mutants, they suggested that the fungal antioxidant system is the molecular target of salicylaldehyde and that vacuolar detoxification plays an important role in fungal resistance to the inhibitory effects of salicylaldehyde. 3.10 Carotenoids from maize and other plants A large number of plant carotenoids have been reported as inhibitors of AF biosynthesis (Norton, 1997 and references therein). Norton (1997) studied the effects of maize carotenoids on AF biosynthesis by A. flavus and found all 11 carotenoids tested except -tocopherol markedly suppressed AFB1 production. The compounds containing the -ionone ring i.e. carotene, lutein and -ionone were the most active carotenoids capable of inhibiting >90% AFB1 production. Exposure of a norsolorinic acid (NOR) accumulating mutant of A. parasiticus SRRC162 to -carotene resulted in production of low levels of both NOR and AF, indicating that a target site(s) of -carotene likely are at early steps of AF pathway before NOR formation. Comparative analysis of chemical structures of tested carotenoids showed the conjugated tail and the double-bond arrangement of the ring to be the determinants of the AF inhibitory activities. Based on the structure/activity data, modification of cell membranes that indirectly affect cytosolic polyketide synthase and specific interaction with hydrophobic domains of AF pathway enzymes were postulated to result in the observed inhibition (Norton, 1997). 3.11 Resistance associated proteins from maize kernel (RAPs) The information derived from genomics, proteomics and metabolomics has provided us a better understanding of how the AF producing fungi survive in the field and how they invade host plants and produce AF (Bhatnagar et al., 2008; Brown et al., 2010; Kim et al., 2007; Rajasekaran et al., 2006). Published studies revealed that plants respond to fungal invasion and infection through: i) producing inhibitors to fungal cell wall degrading enzymes, ii) producing specific inhibitors against fungal growth and/or AF production, iii) producing ROS and stress responsive proteins, iv) increasing lignification and cell wall cross-linking and v) triggering host cell death at the site of infection (Bhatnagar et al., 2008; Liang et al., 2006). Among crops susceptible to AF contamination, maize has been the subject of several studies because of its importance as human staple and as animal feeds worldwide. Natural resistance to AF contamination has been noticed in maize genotypes during field screening. Comparative proteomics studies have identified maize kernel resistance associated proteins (RAPs) as promising breeding markers (Bhatnagar et al., 2008; Brown et al., 2010). RAPs were classified in three major groups including antifungal, storage and stress-responsive proteins. A RAP from maize kernel, the 14 kDa trypsin inhibitor, in resistance to fungal invasion and AF contamination has been confirmed (Brown et al., 2010 and references therein). This trypsin inhibitor indirectly suppresses AF production by inhibiting -amylase of A. flavus, a fungal pathogenesis factor (Chen et al., 1998; Fakhoury & Woloshuk, 1999). Extracellular hydrolases of A. flavus including -amylase are responsible for degrading starch to glucose and maltose used for fungal growth. Fakhoury & Woloshuk (1999) first described Amy1 as -amylase gene of A. flavus and confirmed the role of amylase in AF biosynthesis. They showed that -amylase produced by A. flavus generated sugar concentrations sufficient to induce AF biosynthesis. How different classes of RAPs

294

Aflatoxins – Biochemistry and Molecular Biology

contribute to resistance of maize to AF accumulation is a key question remains to be answered.

4. Microorganisms and their bioactive metabolites Beneficial microorganisms especially bioactive fungi, bacteria and actinomycetes are cell factories that can produce a wide array of biologically active substances inhibitory to AF production. It has been reported that, on average, two or three antibiotics derived from microorganisms enter into the market each year (Clark, 1996). Hundreds of antifungal compounds also have been isolated from different fungi, bacteria and actinomycetes. Terrestrial actinomycetes especially those classified in the genus Streptomyces are rich sources of antifungal and AF inhibitory metabolites (Deshpande et al., 1988). Most recently, the roles of mycoviruses and RNA silencing in relation to AF control have gained special attention (Hammond et al., 2008; Schmidt 2009). 4.1 Bacteria and actinomycetes 4.1.1 Cyclo(L-Leucyl-L-Prolyl); A cyclic dipeptide from Achromobacter xylosoxidans Achromobacter xylosoxidans is a non-fermentative, gram-negative bacillus belonging to the family Alcaligenaceae. It has been associated with a variety of clinical cases ranging from superficial sepsis to potentially fatal nosocomial infections. A. xylosoxidans is a newly emerging microorganism isolated with increased frequency from the lungs of patients with cystic fibrosis, but information about its clinical relevance is limited (Saiman & Siegel, 2004). Yan et al. (2004) reported a new biological activity for an environmental strain of A. xylosoxidans that inhibited AF production by A. parasiticus. The inhibitory metabolite was successfully isolated by a combination of chromatographic techniques and identified as a heat and chemical resistant cyclic dipeptide, "cyclo(L-Leucyl-L-Prolyl)". This compound has also been reported from different organisms including Streptomyces sp., an ascomycete (Rosellinia necatrix), a marine sponge (Rhaphisia pallida) and Halobacillus litoralis, a marine bacterium (Yan et al., 2004 and references therein). By using a tip culture method, the IC50 value of the compound on AFB1 production was determined to be 200 µg/ml. It was inhibitory to A. parasiticus growth at a high concentration of 6000 µg/ml. RT-PCR analyses showed that cyclo(L-Leucyl-L-Prolyl) inhibited AF biosynthesis by repressing transcription of AF pathway genes aflR, hexB, pksL1 and dmtA. The feeding experiment for conversion of sterigmatocystin (ST) to AF in the presence of the compound showed the loss of the most enzymes involved in the pathway from ST to AF. Cyclo(LLeucyl-L-Prolyl) may direct or indirect affect the expression of the pathway regulatory gene, aflR. Further studies are needed in order to elucidate the underlying mechanisms of the inhibition. 4.1.2 Dioctatin A from Streptomyces sp. SA-2581 Dioctatin A was first isolated from Streptomyces sp. SA-2581 by Takeuchi et al. (1991) as an inhibitor of human dipeptidyl peptidase II, a property which accounts for immunosuppressive effects of the compound. Dioctatin A is a white powder with molecular formula of C12H39N3O4, molecular mass of 397.6 Da and a melting point of 263-265 ºC. Yoshinari et al. (2007) demonstrated that Dioctatin A also is a strong inhibitor of AF production by A. parasiticus. AF production was inhibited with an IC50 value at 4.0 µM without any obvious effect on fungal growth. Using RT-PCR, they showed that dioctatin A

Aflatoxins: Mechanisms of Inhibition by Antagonistic Plants and Microorganisms

295

inhibited the transcription of pksA, ver1 and omtA and significantly repressed the pathway regulatory gene, aflR. Besides inhibition in AFB1 and AFG1 production, an efficient suppression of conidiation was observed on solid medium accompanied by a concomitant reduction in the mRNA level of brlA, which encodes a conidiation-specific transcription factor. Based on the data about inhibition of ST biosynthesis and conidia formation in A. nidulans, they proposed that dioctatin A may target the G protein signaling pathway and thus results in inhibition of AF biosynthesis. Dioctatin A may be a good bioactive agent for the control of AF contamination based on several proven benefits including simple structure, no toxicity to mammals, inhibiting AF production in a model infection system on raw peanuts, inhibition of AF and conidiogenesis without affecting fungal growth (lowering the chance for spread of resistance), and targeting only secondary but not primary metabolism. 4.1.3 Aflastatin A from Streptomyces sp. MRI142 Aflastatin A was first isolated from solvent extracts of the mycelial cake of a soil isolate of Streptomyces sp. MRI142 (Ono et al., 1997). Using NMR and chemical degradation analyses, it was revealed that the compound has a skeleton of tetramic acid derivative with a highly oxygenated long alkyl chain (Ono et al., 1998). It is active against different yeasts, mycelial fungi and gram-positive but not gram-negative bacteria (Ono et al., 1997). Aflastatin A completely inhibited AF production by A. parasiticus NRRL2999 in liquid and solid cultures at a concentration of 0.5 µg/ml without affecting fungal growth (Ono et al., 1997). Its inhibitory mechanism was studied by evaluating the effect on AF biosynthetic pathway and glucose metabolism in A. parasiticus (Kondo et al., 2001). Inhibition of NOR production was observed when A. parasiticus ATCC24690 was cultured on potato dextrose agar plates in the presence of 0.1% (v/v) of aflastatin A. Glucose metabolism and ethanol accumulation were accelerated with a marked suppression in transcription of related genes aldA and facA. RT-PCR of the AF biosynthesis genes showed a significant reduction in transcription of pksA, ver1, omtA and aflR when cells were exposed to this compound. Aflastatin A may suppress AF biosynthesis either directly via affecting aflR transcription or indirectly by causing a marked disturbance in the regulatory machinery of carbon metabolism. 4.1.4 Blasticidins A and S from Streptomyces griseochromogenes Blasticidin A, a peptidyl nucleoside antibiotic, was first reported by Fukunaga et al. (1958) as an anti-phytopathogenic substance and its absolute configuration was determined by Sakuda et al. (2007). It is a potent inhibitor of AF biosynthesis by A. parasiticus (Sakuda et al., 2000). Using two-dimensional differential gel electrophoresis (2D-DIGE) Yoshinari et al. (2010) showed that blasticidin A inhibited AF (total of B1 and G1) production and fungal growth with IC50 values of 0.25 and 1.6 µM, respectively. MALDI-TOF MS analysis of protein spots on the 2D-DIGE gel of blasticidin A-treated A. flavus showed decreased amounts of AF biosynthetic enzymes including Vbs, OmtB, OmtA, NorA, Ver-1 and Nor-1 after 36 h treatment. Levels of other proteins with unknown functions were also decreased. It was suggested that protein synthesis in toxigenic fungi maybe the possible target site of blasticidin A. Blasticidin S, another peptidyl nucleoside antibiotic highly similar to blasticidin A, was reported in the same study as an inhibitor of AF production (IC50 = 28 µM) with weak inhibitory effect on fungal growth (IC50 >1000 µM).

296

Aflatoxins – Biochemistry and Molecular Biology

4.2 Mushrooms and microfungi 4.2.1 β-glucans and culture filtrates from Lentinula edodes and Trametes versicolor Mushrooms have received major attention with regard to their biological properties including healing effects against different diseases, antioxidant, anticancer, antiviral, and antibacterial properties and hepatoprotective effects against AF (Zjalic et al., 2006 and references therein). Recently, mushrooms have been explored as potential control agents for AF contamination (Reverberi et al., 2005; Zjalic et al., 2006). Reverberi et al. (2005) concluded that culture filtrate and purified β-glucans from Lentinula edodes, an edible basidiomycetous mushroom native to East Asia, significantly inhibited AF production by A. parasiticus without affecting fungal growth. Their RT-PCR analyses of treated A. parasiticus mycelia showed a delay in activation of AF biosynthetic pathway genes aflR and norA as well as a simultaneous activation of hsf2-like, a transcription factor involved in oxidative stress responses. They suggested that AF production inhibition by β-glucans and culture filtrate of L. edodes resulted from a stimulation of fungal anti-oxidant system that activates antioxidant enzymes such as SOD, catalase and glutathione peroxidase. As a consequence, a delay in AF gene transcription leads to a marked suppression of AF biosynthesis. Mushroom constituents have some advantages over chemicals and plants extracts including low toxicity, simple extraction procedures and easy production on waste materials. Therefore, L. edodes culture filtrate is a promising tool to control pre- and post-harvest AF contamination of crops. Zjalic et al. (2006) examined another industrially important mushroom, Trametes versicolor, for its antifungal activity against an AF producing A. parasiticus. They showed that lyophilized culture filtrates and purified exopolysaccharides of different strains of T. versicolor efficiently inhibited AF (B and G series) production in the range of 40-90% in submerged cultures and on maize and wheat seeds without affecting fungal growth. Antioxidant activity of T. versicolor culture filtrate possibly is associated with β-glucan, a free radical scavenging agent that suppresses AF biosynthesis. RT-PCR analyses of AF biosynthetic genes showed that T. versicolor filtrate also significantly inhibited expression of norA and markedly delayed aflR transcription. 4.2.2 Wortmannin from Penicillium and other microfungi Wortmannin is a furanosteroid fungal metabolite produced by different mycelial fungi especially Penicillium funiculosum and Talaromyces wortmannii (Bräse et al., 2009). It is well known for its biological activity as a specific covalent inhibitor of phosphoinositide 3kinases. These group of enzymes are responsible for regulating various cell survival signaling pathways including growth and proliferation, receptor mediated endocytosis, apoptosis and membrane trafficking in mammalian cells (Shepherd et al., 1988). Recently, Lee et al. (2007) showed that wortmannin at a concentration of 1 µM inhibited fungal growth, asexual sporulation, AF production and expression of AF pathway genes ver1 and nor1. The inhibition on AF biosynthesis appears to interfere the phopsphatidyl inositol 3kinase-mediated signaling pathway similar to that described for mammalian hepatocytes by Rondione et al. (2000). In the model proposed, a cascade of events including blocking of the phopsphatidylinositol 3-kinase activity, inhibition of phosphodiesterase activation, accumulation of cAMP levels to higher than the physiological state, reduction of aflR expression and activity, and reduction of promoter activity of nor1 and ver1 genes occur in wortmannin-treated A. parasiticus which concertedly led to strong inhibition of AF production.

Aflatoxins: Mechanisms of Inhibition by Antagonistic Plants and Microorganisms

297

4.3 dsRNA viruses; RNA silencing as a mechanism for AF suppression Viruses, the fundamental component of life, are involved in modulating intracellular gene activities. They are unique among microorganisms in terms of adaptability, propagation of genomic materials and cellular metabolism. The history of interaction between viruses and aflatoxigenic fungi dates back more than 20 years ago when Schmidt and co-workers (1986) described the effects of viruses on AF production by A. flavus. Transfection experiments with naked and complete dsRNA virus from Penicillium chrysogenum (PcV) which shared similarities in structure and size to dsRNA materials of a non-toxigenic strain of A. flavus resulted in a stable suppression of AF biosynthesis by toxigenic A. flavus. The recent descriptions of RNA interference (Schmidt 2004) and the interaction of Aspergillus mycoviruses with their host via RNA interference (Hammond & Keller, 2008; Hammond et al., 2008) suggest that dsRNA virus from P. chrysogenum may degrades transcripts of AF genes by the RNA interference mechanism (Schmidt et al., 2009). However, further experiments using morphological traits revealed that the PcV-gene suppressing effect on AF biosynthesis is probably nonspecific because it also affected genes involved in both morphogenesis and secondary metabolism (Schmidt, 2004). Schmidt (2009) proposed that suppression of veA gene by PcV-induced siRNAs eventually led to the blocking of AF biosynthesis in the virus transfected A. flavus.

5. Concluding remarks and future prospective Despite the rapid growth of our knowledge in genetics and molecular biology of AF biosynthesis in recent years, little has been documented on how we can practically combat the global problem of AF contamination of crops and agricultural commodities. The information derived from genomic resources such as whole genome sequence and expressed sequence tag (EST) of A. flavus, as well as from proteomics and metabolomics studies will provide us a better understanding of how AF-producing fungi survive in the field and how they invade host plants and produce the carcinogenic AFs. A large number of compounds originated from plants and microorganisms have been proven as strong inhibitors of AF biosynthesis. Recent advances in the identification of the target sites of these inhibitors have shown that they may act via i) interfering with the signal transduction regulatory networks involved in AF gene expression, ii) blocking activities of AF biosynthetic enzymes, iii) down-regulating fungal genes of the oxidative stress defense system that combats metabolic and environmental stressors, iv) inhibiting fungal pathogenesis factors and v) disrupting mitochondrial respiration, a critical process that provides acetyl-CoA for AF biosynthesis. Elucidation of the underlying mechanisms by which plants and microorganisms and their bioactive metabolites affect AF biosynthesis is a major focus in the current molecular biological studies of AF biosynthesis. This endeavor also will advance the knowledge on the complex host plant-toxigenic fungus interactions, which is one of the most important aspects in solving the AF contamination problem.

6. Acknowledgements The authors wish to thank Dr. Kenneth Ehrlich for his kind contribution in preparing Fig. 1.

298

Aflatoxins – Biochemistry and Molecular Biology

7. References Alinezhad, S.; Kamalzadeh, A.; Shams-Ghahfarokhi, M.; Rezaee, M. B.; Jaimand, K.; Kawachi, M.; Zamani, Z.; Tolouei, R. & Razzaghi-Abyaneh, M. (2011). Search for novel antifungals from 49 indigenous medicinal plants: Foeniculum vulgare and Platycladus orientalis as strong inhibitors of aflatoxin production by Aspergillus parasiticus. Annals of Microbiology, doi: 10.1007/s13213-010-0194-1. Allameh, A.; Razzaghi-Abyaneh, M.; Shams, M.; Rezaee, M. B. & Jaimand, K. (2001). Effects of neem leaf extract on production of aflatoxins and activities of fatty acid synthetase, isocitrate dehydrogenase and glutathione S-transferase in Aspergillus parasiticus. Mycopathologia, 154, 79-84. Bakkali, F.; Averbeck, S.; Averbeck, D. & Idaomar, M. (2008). Biological effects of essential oils-A review. Food and Chemical Toxicology, 46, 446-475. Bhatnagar, D. & McCormic, S. P. (1988). The inhibitory effect of neem (Azadirachta indica) leaf extracts on aflatoxin synthesis in Aspergillus parasiticus. Journal of the American Oil Chemists Society, 65, 1166–1168. Bhatnagar, D.; Rajasekaran, K.; Payne, G. A.; Brown, R. L.; Yu, J. & Cleveland, T. E. (2008). The ‘omics’ tools: genomics, proteomics, metabolomics and their potential for solving the aflatoxin contamination problem. World Mycotoxin Journal, 1, 3-12. Bradley, P. (1993). The British Herbal Compendium: Vol. 1: A Handbook of Scientific Information on Widely Used Plant Drugs. British Herbal Medicine Association, London, UK. Bräse, S.; Encinas, A.; Keck, J. & Nising, C. F. (2009). Chemistry and biology of mycotoxins and related fungal metabolites. Chemical Reviews, 109, 3903-3990. Brown, R. L.; Chen, Z. –Y.; Warburton, M.; Luo, M.; Menkir, A.; Fakhoury, A. & Bhatnagar, D. (2010). Discovery and characterization of proteins associated with aflatoxinresistance: Evaluation their potential as breeding markers. Toxins, 2, 919-933. Brown, R. L.; Cotty, P. J. & Cleveland, T. E. (1991). Reduction in aflatoxin content of maize by atoxigenic strains of Aspergillus flavus. Journal of Food Protection, 54, 623-626. Buchanan, R. l. & Lewis, D. F. (1984). Caffeine inhibition of aflatoxin synthesis: Probable site of action. Applied and Environmental Microbiology, 47, 1216-1220. Cai, J.; Zeng, H.; Shima, Y.; Hatabayashi, H.; Nakagawa, H.; Ito, Y.; Adachi, Y.; Nakajima, H. & Yabe, K. (2008). Involvement of the nadA gene in formation of G-group aflatoxins in Aspergillus parasiticus. Fungal Genetics and Biology, 45, 1081-1093. Cary, J. F.; Harris, P. Y.; Molyneux, R. J. & Mahoney, N.E. (2003). Inhibition of aflatoxin biosynthesis by gallic acid. In: Proceedings of the 3rd Fungal Genomics, 4th Fumonisin, and 16th Aflatoxin Elimination Workshop. 13–15 October, 2003, Savannah, GA., p 51. Cary, J. W.; Klich, M. A. & Beltz, S. B. (2005). Characterization of aflatoxin-producing fungi outside of Aspergillus section Flavi. Mycologia 97, 425-432. Chanda, A.; Roze, L. V.; Kang, S.; Artymovich, K. A.; Hicks, G. R.; Raikhel, N. V.; Calvo, A. M. & Linz, J. E. (2009). A key role for vesicles in fungal secondary metabolism. Proceedings of the National Academy of Sciences USA, 106, 19533-19538. Chang, P. –K. & Hua, S. –S. T. (2007). Nonaflatoxigenic Aspergillus flavus TX9-8 competitively prevents aflatoxin accumulation by A. flavus isolates of large and small sclerotial morphotypes. International Journal of Food Microbiology, 114. 275-279.

Aflatoxins: Mechanisms of Inhibition by Antagonistic Plants and Microorganisms

299

Chen, Z. -Y.; Brown, R. L.; Lax, A. R.; Guo, B. Z.; Cleveland, T. E.; & Russin, J. S. (1998). Resistance to Aspergillus flavus in corn kernels is associated with a 14-kDa protein. Phytopathology, 88, 276-281. Clark, A. M. (1996). Natural products as a resource for new drugs, Pharmaceutical Research, 13, 1133-1141. Crowden, R. K.; Harborne, J. B. & Heywood, V. H. (1969). Chemosystematics of the Umbelliferae–A general survey. Phytochemistry, 8, 1963-1984. Dai, J. & Mumper, R. J. (2010). Plant phenolics: extraction, analysis and their antioxidant and anticancer properties. Molecules, 15, 7313-7352. Deshpande, B. S.; Ambedkar, S. S. & Shewale, J. G. (1988). Biologically active secondary metabolites from Streptomyces. Enzyme and Microbial Technology, 10, 455-473. Donner, M.; Atehnkeng, J.; Sikora, R. A.; Bandyopadhyay, R. & Cotty, P. J. (2010). Molecular characterization of atoxigenic strains for biological control of aflatoxins in Nigeria. Food Additives and Contaminants, 27, 576-590. Dorner, J. W. (2004). Biological control of aflatoxin contamination of crops. Journal of Toxicology and Toxin Reviews, 23, 425–450. Ehrlich, K. C.; Scharfenstein, L. L. Jr.; Montalbano, B. G. & Chang, P. K. (2008). Are the genes nadA and norB involved in formation of aflatoxin G1? International Journal of Molecular Sciences, 9, 1717-1729. Fakhoury A. M. & Woloshuk, C. P. (1999). Amy1, the -amylase gene of Aspergillus flavus: involvement in aflatoxin biosynthesis in maize kernels. Phytopathology, 89, 908-914. Franke, R. & Schilcher, H. (2005). Chamomile: Industrial Profiles (Medicinal and Aromatic Plants). CRC Press, Boca Raton, FL, USA. Frisvad, J. C.; Skouboe, P. & Samson, R. A. (2005). Taxonomic comparison of three different groups of aflatoxin producers and a new efficient producer of aflatoxin B1, sterigmatocystin and 3-O-methylsterigmatocystin, Aspergillus rambellii sp. nov. Systematics and Applied Microbiology, 28, 442-453. Fukunaga, K.; Misato, T.; Ishii, I. & Asakawa, M. (1958). Blasticidin, a new antiphytopathogenic fungal substance. Part I. Bulletin of the Chemical Society of Japan, 19, 181–188. Ganzera, M.; Schneider, P. & Stuppner, H. (2005). Inhibitory effects of the essential oil of chamomile (Matricaria recutita L.) and its major constituents on human cytochrome P450 enzymes. Life Sciences, 78, 856–861. Garber, R. K. & Cotty, P. J. (1997). Formation of sclerotia and aflatoxins in developing cotton bolls infected by the S strain of Aspergillus flavus and potential for biocontrol with an atoxigenic strain. Phytopathology, 87, 940–945. Georgianna, D. R. & Payne, G. A. (2009). Genetic regulation of aflatoxin biosynthesis: From gene to genome. Fungal Genetics and Biology, 46, 113-125. Hammond, T. M.; Andrewski, M.D.; Roossninck, M. J. & Keller, N. P. (2008). Aspergillus mycoviruses are targets and suppressors of RNA silencing. Eukaryotic Cell, 7, 350– 357. Hammond, T. M. & Keller, N. P. (2008). RNA silencing in the Aspergilli. Mycology Series, 26, 197–209. Hedayati, M. T.; Pasqualetto, A. C.; Warn, P. A.; Bowyer, P. & Denning, D. W. (2007). Aspergillus flavus: human pathogen, allergen and mycotoxin producer. Microbiology, 153, 1677-1692.

300

Aflatoxins – Biochemistry and Molecular Biology

Holmes, R. A.; Boston, R. S. & Payne, G.A. (2008). Diverse inhibitors of aflatoxin biosynthesis. Applied Microbiology and Biotechnology, 78, 559-572. Hua, S. -S. T. (2004). Application of a yeast, Pichia anomala strain WRL-076 to control Aspergillus flavus for reducing aflatoxin in pistachio and almond. Bulletin of International Organization for Biological and Integrated Control of Noxious Animals and Plants 27, 291–294. Hua, S. –S. T.; Grosjean, O.-K. & Baker, J. L. (1999). Inhibition of aflatoxin biosynthesis by phenolic compounds. Letters in Applied Micobiology, 29, 289-291. Jayashree, T. & Subramanyam, C. (2000). Oxidative stress as a prerequisite for aflatoxin production by Aspergillus parasiticus. Free Radical Biology & Medicine, 29, 981-985. Keller, N. P.; Kantz, N. J. & Adams, T. H. (1994). Aspergillus nidulans verA is required for production of the mycotoxin sterigmatocystin. Applied and Environmental Microbiology, 60, 1444-1450. Keller, N. P.; Segner, S.; Bhatnagar, D. & Adams, T. H. (1995). stcS, a putative P-450 monooxygenase, is required for the conversion of versicolorin A to sterigmatocystin in Aspergillus nidulans. Applied and Environmental Microbiology, 61, 3628-3632. Kim, J. H.; Campbell, B. C.; Yu, J.; Mahoney, N.; Chan, K. L.; Molyneux, R. J.; Bhatnagar, D. & Cleveland, T.E. (2005). Examination of fungal stress response genes using Saccharomyces cerevisiae as a model system: targeting genes affecting aflatoxin biosynthesis by Aspergillus flavus Link. Applied Microbiology and Biotechnology, 67, 807–815. Kim, J. H.; Mahoney, N.; Chan, K. L.; Molyneux, R. & Campbell, b. C. (2006) Controlling Food-contaminating fungi by targeting antioxidant stress-response system with natural phenolic compounds. Applied Microbiology and Biotechnology, 70, 735-739. Kim, J. H.; Yu, J.; Mahoney, N.; Chan, K. L.; Molyneux, R. J.; Varga, J.; Bhatnagar, D.; Cleveland, T. E.; Nierman, W. C. & Campbell, B. C. (2008). Elucidation of the functional genomics of antioxidant-based inhibition of aflatoxin biosynthesis. International Journal of Food Microbiology, 122, 49-60. Kim, J. H.; Campbell, B. C.; Mahoney, N.; Chan, K. L. & Molyneux, R. J. (2010). Chemosensitization of aflatoxigenic fungi to antimycin A and strobilurin using salicylaldehyde, a volatile natural compound targeting cellular antioxidant system. Mycopathologia, doi: 10.1007/s11046-010-9356-8. Kim, Y. H.; Woloshuk, C. P.; Cho, E. H.; Bae, J. M.; Song, Y.-S. & Huh, G. H. (2007) Cloning and functional expression of the gene encoding an inhibitor against Aspergillus flavus -amylase, a novel seed lectin from Lablab purpureus (Dolichos lablab).Plant Cell Reports, 26, 395-405. Kondo, T., Sakurada, M., Okamoto, S., Ono, M., Tsukigi, H., Suzuki, A., Nagasawa, H. & Sakuda, S. (2001). Effects of aflastatin A, an inhibitor of aflatoxin production, on aflatoxin biosynthetic pathway and glucose metabolism in Aspergillus parasiticus. The Journal of Antibiotics (Tokyo), 54, 650–657. Kong, C.; Hu, F.; Xu, X.; Liang, W. & Zhang, C. (2004). Allelopathic plants. XV. Ageratum conyzoides L. Allelopathy Journal, 14, 1–12. Korkina, L. G. (2007). Phenylpropanoids as naturally occurring antioxidants: from plant defense to human health. Cellular and Molecular Biology, 53, 15-25.

Aflatoxins: Mechanisms of Inhibition by Antagonistic Plants and Microorganisms

301

Lee, J. –W.; Roze, L. V. & Linz, J. E. (2007). Evidence that a wortmannin-sensitive signal transduction pathway regulates aflatoxin biosynthesis. Mycologia, 99, 562-568. Liang, X. Q.; Luo, M. & Guo, B. Z. (2006). Resistance mechanisms to Aspergillus flavus infection and aflatoxin contamination in peanut (Arachis hypogaea). Plant Pathology Journal, 5, 115-124. Lima, C.F.; Fernandes-Ferreira, M. & Pereira-Wilson, C. (2006). Phenolic compounds protect HepG2 cells from oxidative damage: relevance of glutathione levels. Life Sciences, 79, 2056–2068. MacRae, W. D. & Towers, G. H. N. (1984). Biological activities of lignans. Phytochemistry 23, 1207-1220. Mahoney N, Molyneux RJ (2004) Phytochemical inhibition of aflatoxigenicity in Aspergillus flavus by constituents of walnut (Juglans regia). Journal of Agricultural and Food Chemistry, 52, 1882–1889. Martinez, V.; Barbera, O.; Sanchez-Parareda, J. & Marco, J. A. (1987). Phenolic and acetylenic metabolites from Artemisia assoana. Phytochemistry, 26, 2619–2624. Narasaiah, K. W.; Sashidhar, R. B. & Subramanyam, C. (2006) Biochemical analysis of oxidative stress in the production of aflatoxin and its precursor intermediates. Mycopathologia, 162, 179–189. Newall, C. A.; Anderson, L. A. & Phillipson, J. D. (1996). Herbal Medicines: a guide for healthcare professionals. Pharmaceutical Press, London. Nogueira, J. H. C.; Gonçalez, E.; Galleti, S. R.; Facanali, R.; Marques, M. O. M. & Felício, J. D. (2010) Ageatum conyzoides essential oil as aflatoxin suppressor of Aspergillus flavus. International Journal of Food Microbiology, 137, 55-60. Norton, R. A. (1997). Effect of carotenoids on aflatoxin B1 synthesis by Aspergillus flavus. Phytopathology, 87, 814-821. Ono, M.; Sakuda, S.; Suzuki, A. & Isogai, A. (1997). Aflastatin A, a novel inhibitor of aflatoxin production by aflatoxigenic fungi. The Journal of Antibiotics (Tokyo), 50, 111–118. Ono, M.; Sakuda, S.; Ikeda, H.; Furihata, K.; Nakayama, J.; Suzuki, A. & Isogai, A. (1998). Structures and biosynthesis of aflastatins: novel inhibitors of aflatoxin production by Aspergillus parasiticus. The Journal of Antibiotics (Tokyo), 51, 1019–1028. Pildain, M. B.; Frisvad, J. C.; Vaamonde, G.; Cabral, D.; Varga, J. & Samson, R. A. (2008). Two novel aflatoxin-producing Aspergillus species from Argentinean peanuts. International Journal of Systematic and Evolutionary Microbiology, 58, 725-735. Rajasekaran, K.; Cary, J. W. & Cleveland, T. E. (2006) Prevention of preharvest aflatoxin contamination through genetic engineering of crops. Mycotoxin Research, 22, 118124. Razzaghi-Abyaneh, M,; Allameh, A,; Tiraihi, T,; Shams-Ghahfarokhi, M. & Ghorbanian, M. (2005). Morphological alterations in toxigenic Aspergillus parasiticus exposed to neem (Azadirachta indica) leaf and seed aqueous extracts. Mycopathologia, 159, 565570. Razzaghi-Abyaneh, M.; Yoshinari, T.; Shams-Ghahfarokhi, M.; Rezaee, M. B.; Nagasawa, H. & Sakuda, S. (2007). Dillapiol and apiol as specific inhibitors of the biosynthesis of aflatoxin G1 in Aspergillus parasiticus. Bioscience, Biotechnology and Biochemistry, 71, 2329-2332.

302

Aflatoxins – Biochemistry and Molecular Biology

Razzaghi-Abyaneh, M.; Shams-Ghahfarokhi, M,; Yoshinari, T,; Rezaee, M. B,; Nagasawa, H. & Sakuda, S. (2008). Inhibitory effects of Satureja hortensis L. essential oil on growth and aflatoxin production by Aspergillus parasiticus. International Journal of Food Microbiology, 123, 228-233. Razzaghi-Abyaneh, M.; Shams-Ghahfarokhi, M.; Rezaee, M. B.; Jaimand, S.; Alinezhad, S.; Saberi, R. & Yoshinari, T. (2009). Chemical composition and antiaflatoxigenic activity of Carum carvi L., Thymus vulgaris and Citrus aurantifolia essential oils. Food Control, 20, 1018-1024. Razzaghi-Abyaneh, M.; Shams-Ghahfarokhi, M.; Rezaee, M. B. & Sakuda, S. (2010). Natural Aflatoxin Inhibitors From Medicinal Plants, In: Mycotoxins in Food, Feed and Bioweapons, Rai, M. & Varma, A. (ed.), 329-352, Springer-Verlag, Berlin, Heidelberg. Reverberi, M.; Fabbri, A. A.; Zjalic, S.; Ricelli, A.; Punelli, F. & Fanelli, C. (2005). Antioxidant enzymes stimulation in Aspergillus parasiticus by Lentinula edodes inhibits aflatoxin production. Applied Microbiology and Biotechnology, 69, 207–215. Reverberi, M.; Zjalic, s.; Ricelli, A.; Punelli, F.; Camera, E.; Fabbri, C.; Picardo, M.; Fanelli, C. & Fabbri, A. A. (2008). Modulation of antioxidant defence in Aspergillus parasiticus is involved in aflatoxin biosynthesis: a role for the ApyapA gene. Eukaryotic Cell, 7, 988-1000. Rondinone, C. M.; Carvalho, E.; Rahn, T.; Manganiello, V. C.; Degerman, E. & Smith, U. P. (2000). Phosphorylation of PDE3B by phosphatidylinositol 3-kinase associated with the insulin receptor. Journal of Biological Chemistry, 275, 10093–10098. Saiman, L. & Siegel J. (2004). Infection control in cystic fibrosis. Clinical Microbiology Reviews, 17, 57–71. Sakuda, S.; Ono, M.; Ikeda, H.; Nakamura, T.; Inagaki, Y.; Kawachi, R.; Nakayama, J.; Suzuki, A.; Isogai, A. & Nagasawa, H. (2000a). Blasticidin A as an inhibitor of aflatoxin production by Aspergillus parasiticus. The Journal of Antibiotics (Tokyo), 53, 1265–1271. Salamon, I. (1992). Chamomile, a medicinal plant. The Herb, Spice, and Medicinal Plant Digest, 10, 1–4. Samson, A. R., Hoekstra, E. S.; Frisvad, J. C. & Filtenborg, O. (2000). Introduction to Food and Airborne Fungi, (Sixth edn.), CBS-Utrecht, The Netherlands. Samuelsson, G. (2004). Drugs of Natural Origin: a Textbook of Pharmacognosy, 5th Swedish Pharmaceutical Press, Stockholm. Schmidt, F. R.; Davis, N. D.; Diener, U. L. & Lemke, P. A. (1983). Cycloheximide induction of aflatoxin synthesis in a nontoxigenic strain of Aspergillus flavus. Nature Biotechnology, 1, 794–795. Schmidt, F. R.; Lemke, P. A. & Esser, K. (1986). Viral influences on aflatoxin formation by Aspergillus flavus. Applied Microbiology and Biotechnology, 24, 248–258. Schmidt, F. R. (2004). RNA interference detected 20 years ago? Nature Biotechnology, 22, 267– 268. Schmidt, F. R. (2009). The RNA interference-virus interplay: tools of nature for gene modulation, morphogenesis, evolution and a possible mean for aflatoxin control. Applied Microbiology and Biotechnology, 83, 611-615. Shepherd, P. R.; Withers, D. J. & Siddle, K. (1988). Phosphoinositide 3-kinase: the key switch mechanism in insulin signaling. Biochemical Journal, 333, 471–490.

Aflatoxins: Mechanisms of Inhibition by Antagonistic Plants and Microorganisms

303

Takeuchi, T.; Aoyanagi, T.; Okami, Y.; Osanawa, H.; Iinuma, H. & Ogawa, K. (1991). Novel aminopeptidase-inhibiting dioctatins and their manufacture with Streptomyces species. Japanese Patent JP 03077857 A2 19910403 Heisei. Tolouee, M.; Alinezhad, S.; Saberi, R.; Eslamifar, A.; Zad, S. J.; Jaimand, K.; Taeb, J.; Rezaee, M. B.; Kawachi, M.; Shams-Ghahfarokhi, M. & Razzaghi-Abyaneh, M. (2010). Effect of Matricaria chamomilla L. flower essential oil on the growth and ultrastructure of Aspergillus niger van Tieghem. International Journal of Food Microbiology, 139, 127-133. Trail, F.; Mahanti, N. & Linz, J. (1995). Molecular biology of aflatoxin biosynthesis. Microbiology, 141, 755-765. Udwary, D. W.; Casillas, L. K. & Townsend, C. A. (2002). Synthesis of 11-hydroxyl Omethylsterigmatocystin and the role of a cytochrome P-450 in the final step of aflatoxin biosynthesis. Journal of the American Chemical Society, 124, 5294-5303. Varga, J.; Frisvad, J. C. & Samson, R. A. (2010). A reappraisal of fungi producing aflatoxins. World Mycotoxin Journal, 2, 263-277. Wen, Y.; Hatabayashi, H.; Arai, H.; Kitamoto, H. K. & Yabe, K. (2005). Function of the cypX and moxY genes in aflatoxin biosynthesis in Aspergillus parasiticus. Applied and Environmental Microbiology, 71, 3192-3198. Yabe, K.; Nakamura, M. & Hamasaki, T. (1999). Enzymatic formation of G-group aflatoxins and biosynthetic relationship between G- and B-group aflatoxins. Applied and Environmental Microbiology, 65, 3867-3872. Yaguchi, A.; Yoshinari, T.; Tsuyuki, R.; Takahashi, H.; Nakajima, T.; Sugita-Konishi, Y.; Nagasawa, H. & Sakuda, S. (2009). Isolation and identification of precocenes and piperitone from essential oils as specific inhibitors of trichothecene production by Fusarium graminearum. Journal of Agricultural and Food Chemistry, 57, 846-851. Yan, P. –S.; Song, Y.; Sakuno, E.; Nakajima, H.; Nakagawa, H. & Yabe, K. (2004). Cyclo(LLeucyl-L-Prolyl) produced by Achromobacter xylosoxidans inhibits aflatoxin production by Aspergillus parasiticus. Applied and Environmental Microbiology, 70, 7466-7473. Yoshinari, T.; Akiyama, T.; Nakamura, K.; Kondo, T.; Takahashi, Y.; Muraoka, Y.; Nonomura, Y.; Nagasawa, H. & Sakuda, S. (2007). Dioctatin A is a strong inhibitor of aflatoxin production by Aspergillus parasiticus. Microbiology, 153, 2774-2780. Yoshinari, T.; Yaguchi, A.; Takahashi-Ando, N.; Kimura, M.; Takahashi, H.; Nakajima, T.; Sugita-Konichi, Y.; Nagasawa, H. & Sakuda, S. (2008). Spiroethers of German chamomile inhibit production of aflatoxin G and trichothecene mycotoxin by inhibiting cytochrome P450 monooxygenases involved in their biosynthesis. FEMS Microbiology Letters, 284, 184-190. Yoshinari, T.; Noda, Y.; Yoda, K.; Sezaki, H.; Nagasawa, H. & Sakuda, A. (2010). Inhibitory activity of blasticidin A, a strong aflatoxin production inhibitor, on protein synthesis of yeast: selective inhibition of aflatoxin production by protein synthesis inhibitors. The Journal of Antibiotics, 63, 309-314. Yu, J.; Chang, P. -K.; Cary, J. W.; Bhatnagar, D. & Cleveland, T. E. (1997). avnA, a gene encoding a cytochrome P-450 monooxygenase, is involved in the conversion of averantin to averufin in aflatoxin biosynthesis in Aspergillus parasiticus. Applied and Environmental Microbiology, 63, 1349-1356. Zeng, H.; Hatabayashi, H.; Nakagawa, H.; Cai, J.; Suzuki, R.; Sakuno, E.; Tanaka, T.; Ito, Y.; Ehrlich, K. C.; Nakajima, H. & Yabe, K. (2011). Conversion of 11-hydroxy-Omethylsterigmatocystin to aflatoxin G1 in Aspergillus parasiticus. Applied Microbiology and Biotechnology. Doi: 10.1007/s00253-010-2999-z.

304

Aflatoxins – Biochemistry and Molecular Biology

Zeringue, H. J. Jr. & Bhatnagar, D. (1990). Inhibition of aflatoxin production in Aspergillus flavus infected cotton bolls after treatment with neem (Azadirachta indica) leaf extracts. Journal of the Americal Oil Chemists Society, 67, 215–216. Zjalic, S.; Reverberi, M.; Ricelli, A.; Mario-Granito, V.; Fanelli, K. & Fabbri, A. A. (2006). Trametes versicolor: A possible tool for aflatoxin control. International Journal of Food Microbiology, 107, 243-249.

15 The Evolutionary Dynamics in the Research on Aflatoxins During the 2001-2010 Decade Martín G. Theumer and Héctor R. Rubinstein Centro de Investigaciones en Bioquímica Clínica e Inmunología (CIBICI), CONICET, Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Argentina 1. Introduction The interest on aflatoxins began in the late fifties and the early sixties, after more than 100 000 young turkeys’ deaths were registered in the course of a few months in 1960 on poultry farms in England. Since the deaths were caused by an apparently new disease, it was termed "Turkey X disease". Shortly after, it was observed that the disease also affected ducklings and young pheasants with high rates of mortalities. The disease was initially suspected to be induced by toxins of fungal origin, and later was proved to be caused by aflatoxins, fungal secondary metabolites synthesized by toxigenic stocks of Aspergillus spp. This discovery has led to a growing awareness of the potential hazards of these substances as potential inducers of illnesses and even death in humans and other mammals (Kensler et al., 2011). Thus, the “Turkey X disease” outbreak is widely considered as the initial step in the era of the aflatoxins research. The genus Aspergillus is an extremely common contaminant in stored products in tropical and subtropical regions; mainly grains, nuts and spices, and several Aspergillus species are frequently involved in its decomposition. These molds, and consequently the food contamination with mycotoxins that they synthesize, are ubiquitous in warm regions. Despite this, because the cold-climate countries import grain of geographical areas with tropical and subtropical climates, aflatoxins have gained importance worldwide. Moreover, nowadays the technological processes of food production fail to completely eliminate aflatoxins and therefore are part of food for humans and farm animals, thus favoring the diet-mediated intake of aflatoxins (Park, 2002). The scientific literature on aflatoxins includes more than 8000 research articles, and the toxicological data regarding these compounds led to the International Agency for Research on Cancer to classify 1993 the naturally occurring aflatoxins as Group 1 (carcinogenic to humans), and the aflatoxin M1 (AFM1, an oxidative metabolic product of AFB1) as Group 2B (possibly carcinogenic to humans) (IARC, 1993). In this chapter it is attempted to describe somehow the information generated in the aflatoxins field in the past decade on the basis of an exhaustive categorization of the publications retrieved by entering the keyword “aflatoxin” in the PubMed search engine

306

Aflatoxins – Biochemistry and Molecular Biology

(http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed) of the United States National Library of Medicine web page, a database of biomedical literature widely used around the world. The initial searching criteria included all the articles published (print or electronic) between 2001/01/01 and 2010/12/31.

2. Quantitative characterization of the research on aflatoxins through the 2001-2010 period 2.1 Number of publications Two thousands two hundreds and six results were retrieved by inserting the searching criteria specified above. These results were then further classified according to the year of publication (print or electronic), in order to evaluate the behavior of the research on the aflatoxins area throughout the decade. The figure 1 shows the evolution of the number of annual contributions.

Fig. 1. The behavior in the research on aflatoxins in the 2001-2010 decade, assessed by the number of publications by year. The average number of contributions in the full period was 220 articles by year. Assessed by the number of annual publications, the research on the aflatoxins field decreased annually from years 2001 through 2004; however this tendency was reverted according to the number of contributions registered in 2005, when the interest in the aflatoxins research increased till to the end of the decade, with 267 contributions registered in 2010.

The Evolutionary Dynamics in the Research on Aflatoxins During the 2001-2010 Decade

307

2.2 Type of articles The PubMed search engine also offers the possibility of filtering the requested information according to the type of contribution. Thus the searching results may include only clinical trials (phases I to IV), letters, original or corrected and republished journal articles, reviews, case reports, or congresses, for instance. This tool allows a fast characterization of the type of contribution on a specific issue. With regards to the specific field of the aflatoxins, the main publication type was original journal articles, as depicted in figure 2. The number of reviews was fluctuating in the 2001-2010 decade, and the number of journal articles published in the period showed a similar behavior to that of the total annual contributions.

Fig. 2. The behavior in the research on aflatoxins in the 2001-2010 decade, assessed by the annual number of every main type of contribution. The case reports are of special interest in mycotoxicology, since they may provide evidence for the linking of symptoms with diseases induced by fungal metabolites, or even may introduce new insights on the possible mechanisms of pathogenesis occurring in the natural poisonings with fungal toxins, for instance. A total of 317 cases of acute aflatoxicosis were reported in Kenya by 20 July 2004, with a case fatality rate of 39%. The 2004 outbreak resulted from widespread aflatoxin contamination of locally grown maize, which occurred during storage of the maize under damp conditions (Probst et al., 2007). Mwanda and co-workers (2005) reported a 17-year-old schoolboy case of aflatoxicosis, highlighting the salient clinical findings in order to increase the index of suspicion, enhance early diagnosis and improve management of such cases.

308

Aflatoxins – Biochemistry and Molecular Biology

Case reports of probable aflatoxicosis in animals were also informed in 2004 and 2006. Osman et. al (2004) reported the death of twenty young female adult one-humped racing camels (Camelus dromedarius) kept in camps scattered outside Al Ain city and aged between 3- and 6-years-old, after a short clinical illness. On the other hand, in the USA more than 100 dogs apparently died as a result of the toxic effects of contaminated products, as it was suggested by medical history, clinical signs, progression of disease, and necropsy findings (Stenske et al., 2006). Abstracts of Congresses such as the 3rd Fungal Genomics, 4th Fumonisin, and 16th Aflatoxin Elimination Workshops, 2003, Savannah, Georgia, USA; the Workshop on Aflatoxin and fumonisin elimination and fungal genomics, 2002, San Antonio, Texas, USA; and the Aflatoxin/Fumonisin Elimination and Fungal Genomics Workshops, 2001, Phoenix, Arizona, USA; were also published in the 2001-2010 decade. 2.3 Main contributors A further analysis of journal articles and reviews retrieved by the PubMed search engine was carried out in order to determine the countries with a mayor contribution to the aflatoxins research field, and the results are depicted in figs. 3 and 4.

Fig. 3. Main contributor countries in the aflatoxins research, according to the number of articles published in the 2001-2010 decade. According to the information provided by PubMed, the contributions in the research field of aflatoxins in the 2001-2010 decade were carried out in the following countries, alphabetically listed: Albania, Algeria, Argentina, Australia, Austria, Bangladesh, Belgium, Benin, Botswana, Brazil, Brunei, Bulgaria, Burundi, Cameroon, Canada, China, Colombia, Croatia, Cuba, Czech Republic, Denmark, Ecuador, Egypt, Ethiopia, Finland, France, The Gambia, Germany, Ghana, Greece, Hong Kong, Hungary, India, Indonesia, Iran, Iraq, Ireland, Israel, Italy, Japan, Kenya, North Korea, South Korea, Kuwait, Lebanon, Malawi, Malaysia, Mexico, Morocco, Nepal, Netherlands, New Zealand, Nigeria, Norway, Oman, Pakistan, Philippines, Poland, Portugal, Qatar, Romania, Russia, Samoa, Saudi Arabia, Serbia and Montenegro, Sierra Leone, Slovakia, Slovenia, South Africa, Spain, Sri Lanka, Sudan, Sweden, Switzerland, Taiwan, Tanzania, Thailand, Trinidad and Tobago, Tunisia, Turkey, Uganda, Ukraine, United Arab Emirates, United Kingdom, United States, Venezuela, Vietnam, Zambia.

The Evolutionary Dynamics in the Research on Aflatoxins During the 2001-2010 Decade

Fig. 4. The dynamic of the five main contributor countries in the aflatoxins research, according to the number of articles published by year in the 2001-2010 decade.

309

310

Aflatoxins – Biochemistry and Molecular Biology

The United States of America did the main contribution of original journal articles and reviews published in the aflatoxins research field in the whole period (fig. 3), which was related to highest annual contributions of this country from 2001 to 2010 (fig. 4). China, India, Japan and Italy were also quantitatively important contributors in the full period, with 6.53, 5.85, 5.58 and 5.49% of the total publications, respectively. These five countries contributed with more than a half of the articles related to aflatoxins within the decade. Detailed information on the top-five countries with the highest annual number of publications is shown in fig. 4.

3. Qualitative characterization of the research on aflatoxins through the 2001-2010 period 3.1 General overview The articles related to aflatoxins in the 2001-2010 decade were further classified qualitatively according to the focusing of the work into the following five main sub-areas of interest: i. those oriented in the study of the plants commonly infected by aflatoxin-producing Aspergillus spp.; ii. those where the main objective was to elucidate some issue related to the fungal physiology and its relationship with the environment; iii. those where the interest was in the strategies for preservation and prevention of food contamination with aflatoxins; iv. those focused in the elucidation of the physiological and biochemical disturbances associated with the exposure of cells/organisms to the aflatoxins, and the potential exogenous interventions to prevent them; v. those concerning to the analytical methods commonly applied to detect and quantify aflatoxins in commodities or its by-products, for instance. The fig. 5 shows the percentage of articles focused in every sub-area mentioned above. More than a half of the publications in the 2001-2010 decade were oriented on topics related to the toxicology of aflatoxins. The same percentage of publications was focused in the fungal physiology and its relationship with the environment, and in the strategies for preservation and prevention of food contamination with aflatoxins. The minor proportion of the total contributions were concerning to the plants commonly infected by aflatoxinproducing Aspergillus spp.

Plants, 2% Methods, 14%

Fungus, 16%

Foods, 16%

Toxicology, 52% Fig. 5. Distribution of the total contributions in the decade into the main five focusing subareas in the research on aflatoxins.

311

The Evolutionary Dynamics in the Research on Aflatoxins During the 2001-2010 Decade

The same classification of the articles into the five sub-areas of interest was carried out in order to identify possible inter-annuals variations in the percentages of articles in every subarea. The results of this categorization are shown in the table 1. Year 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010

Plants 3,6 2,5 0,5 1,7 1,9 3,3 1,8 1,3 1,2 1,9

Fungi 10,0 13,2 13,4 19,1 17,2 14,0 21,0 18,6 18,2 12,5

Sub-area of interest Food Toxicology 15,9 58,2 18,3 57,9 16,0 62,9 12,7 56,1 17,6 51,1 17,6 52,0 15,6 42,4 17,7 48,9 14,9 43,0 17,4 50,8

Methods 12,3 8,1 7,2 10,4 12,2 13,1 19,2 13,5 22,7 17,4

Table 1. Annual distribution of the contributions focusing into the main five sub-areas of interest in the research on aflatoxins. The data are expressed as percentages of the total number of publications in the specified year.

The outstanding finding of this classification was the increasing proportion of the publicationsin the second part of the decade focusing in different aspects of the analytical methods. This could be related at least in part, to the increasing demand of new methods to detect simultaneously co-contamination of foods with several micotoxins, which also should be ideally feasible of applying to different matrices. 3.2 Approaches in the articles regarding to plants Most of the work carried out in this sub-area aimed to pinpoint the biochemical basis of the susceptibility or resistance of plants to their infection with aflatoxin-producing fungus, and its subsequent contamination with this mycotoxin. Some examples of the advances in this area are mentioned below. Baker et al. (2009a) used a proteomic approach in order to identify proteins that may be associated with the resistance of maize to the fungus. The authors identified a higher expression of a protein (ZmCORp) in maize resistant to A. flavus infection. The fungistatic activity exhibited by this protein was related with its inhibitory effect on the conidia germination and mycelia growth, concluding that ZmCORp may play an important role in enhancing kernel resistance to A. flavus infection and aflatoxin accumulation. Trypsin inhibitors may also play a role in the susceptibility/resistance of maize to Aspergillus spp. infection. Baker et al. (2009b) observed that an overexpressed trypsin inhibitor reduced the germination of conidia as well as the mycelia growth from several maize pathogens, although the effect of this trypsin inhibitor on fungal growth was weaker than the previously reported for other trypsin inhibitors. A proteomic approach performed by Chen and co-workers (2006) led to the identification of a pathogenesis-related protein (PR-10) which is expressed fivefold higher in maize resistant lines. It was also observed that the expression of this protein was induced upon A. flavus infection in a resistant but not in a susceptible genotype, thus suggesting the participation of this protein in kernel resistance to the infection by A. flavus.

312

Aflatoxins – Biochemistry and Molecular Biology

In a recent contribution, Gao and collaborators (2009) studied the potential interference of plant-derived oxylipins (a set of substances produced by the oxidation of polyunsaturated fatty acids by plant and fungal lipoxygenases) in the fungal development and in the secondary metabolism. The authors found contrasting results with different fungal pathogens, suggesting that the modulation of the plant-pathogen interactions are pathogen specific. A novel PLD gene, encoding a putative phospholipase D, was identified in Arachis hypogea by Gu et al. (2006). The authors concluded that this PLD may be involved in drought sensitivity and tolerance response, and they also suggest the study of the PLD gene expression as a tool in germplasm screening for drought tolerance. 3.3 Approaches in the articles regarding to fungus Several aspects were evaluated with regards to the fungi involved in the aflatoxins biosynthesis, including the molecular characterization of isolates in order to elucidate their phylogenetic relationships, the fungal biology and the genetic regulation leading to the mycotoxins production, the biochemical basis underlying the fungal reproduction, for instance. According to the publications registered in the 2001-2010 decade, much of the researchers’ attention was on the identification of physical, chemical or biological conditions that ultimately reduce the fungal-induced food alterations and their contamination with aflatoxins. A wide spectrum of physical, chemical and biological agents was tested in attempts to solve such problems. The essential oils from plants are complex mixtures of substances that in some cases exert strong biological activities against microorganisms. This property attracts the attention of researchers looking for new strategies of chemoprevention of the food contamination with mycotoxins. Gandomi et al. (2010) tested the effects of Zataria multiflora Boiss. essential oil (EO) on A. flavus. The EO suppressed the size of colonies as well as the fungal esporulation. Electronic microscopy revealed morphological alterations ranging from loss of turgidity and uniformity of mycelia at low concentrations of EO to evident destruction of the hyphae at higher concentration of EO, mechanisms that could be involved in the fungal growth and in the biosynthesis of aflatoxins. On the other hand, nine different oils were evaluated by Juglal and Govinden (2002) on the growth of A. parasiticus and Fusarium moniliforme. The highest growth inhibitory activity was found when clove oil (eugenol) was used, followed by cinnamon (cinnamic aldehyde), oregano (thymol and carvacol) and mace oils (myristin). Neem and eucalyptus oil (cineole) did not affect the fungal growth. Then, commonly occurring mycotoxigenic fungi could be controlled with clove oil (eugenol). In other study, Kumar et al. (2007) observed that the EO extracted from the leaves of Chenopodium ambrosioides Linn. completely inhibited the mycelial growth and the aflatoxin synthesis, also exerting a broad fungitoxic spectrum against several toxigenic and nontoxigenic funguses. Reverberi and co-workers (2005) studied the mechanisms underlying the aflatoxininhibiting effect of the Lentinula edodes culture filtrates. The authors reported that L. edodes lyophilised filtrates stimulate A. parasiticus anti-oxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase) and aflatoxin inhibition was better correlated with betaglucan content than with anti-oxidant activity of the filtrates. Several studies were performed with chemical substances purified from different sources. For instance, Cleveland et al. (2009) studied the effects of volatile compunds (including

The Evolutionary Dynamics in the Research on Aflatoxins During the 2001-2010 Decade

313

aldehydes, alcohols, ketones, and furans) produced upon exposure of soybean homogenates to lipase, on the A. flavus growth and aflatoxin production. They found that aldehydes inhibited up to 100% of the observed fungal growth and AFB1 production. Goncalez and co-workers (2001) studied the effects of four biflavonoids isolated from Ouratea species on the A. flavus development and production of aflatoxins. These authors observed that the four biflavonoids showed inhibitory activity on aflatoxin B1 and B2 production, without alterations in the mycelial growth. Analytical and industrial grade food additives effects on toxigenic Aspergillus were also tested, in order to identify a potential use as preservatives in foods. Passone et al. (2007) assayed the effects of three-food grade antioxidants: butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and propyl paraben (PP) to prove their fungitoxic effect on Aspergillus section Flavi strains. The authors concluded that idustrial grade antioxidant mixtures could be used for controlling the growth of mycotoxigenic species. The biological control is a promising field to solve, at least in part, the problem of food contamination with aflatoxins. Several microorganisms could be use for this purpose, including bacteria and fungus. Kong et al. (2010) evaluated a strain of marine Bacillus with regards to its activity in reducing postharvest decay of peanut kernels caused by A. flavus, and demonstrated that this marine bacterium could be used as a biocontrol agent against postharvest fungal disease. Other bacteria such as Bacillus subtilis (Mohammadipour et al., 2009), isolations from almond flowers and mature nut fruits (Palumbo et al., 2006), and the Lactobacillus plantarum ATCC 8014 (Xu et al., 2003) were evaluated for their potential biocontrolling activities against aflatoxin-producing Aspergillus. Studies with nontoxigenic strains of A. flavus and A. parasiticus separately and in combination were also conducted to determine the efficacy for reducing aflatoxin contamination in corn (Dorner, 2009) and peanuts (Dorner & Horn, 2007). Whereas the results of some of these experiments indicated that the treatment with nontoxigenic A. flavus strains could be more effective than with the A. parasiticus strains, others results were inconclusive. Penna & Etcheverry (2006) observed that the interaction of Kluyveromyces spp. with Aspergillus aflatoxigenic strains induced changes on conidia germination, mycelial growth and aflatoxin B1 accumulation. Since biological control can potentially have positive and negative effects on biodiversity, additional efforts are needed to clearly determine a minimum impact on nature by the introduction of such biological control strategies. 3.4 Approaches in the articles regarding to foods Most of the articles classified within this category were surveys on the mycoflora and contamination of foods with aflatoxins. The fungal and aflatoxins contamination was reported in rice in Turkey (Aydin et al., 2010); in maize from south-western Nigeria (Bankole & Mabekoje, 2004), Brazil (Rocha et al., 2009) and northern Italy (Pietri et al., 2004); in domestic and imported beers in Canada (Mably et al., 2005); and in peanuts in the Cote d'Ivoire (Sangare-Tigori et al., 2006), between others. Since contamination of grains with mycotoxins is not efficiently eliminated by most of the food processing operations such as sorting, trimming, cleaning, milling, brewing, cooking, baking, frying, roasting, canning, flaking, alkaline cooking, nixtamalization, and extrusion;

314

Aflatoxins – Biochemistry and Molecular Biology

the fungal toxins are commonly found in grain by-products. Thus, the design of new technological food-processing methods that completely eliminate the mycotoxins is a field of growing interest for researchers worldwide. Castells et al. (2008) studied the distribution of aflatoxins in various corn processed fractions during industrial cornflake processing. These authors observed that the application of dry milling of corn led to a heterogeneous distribution of the two groups of mycotoxins in the different parts of the grain, with increased levels in fractions processed from outer layers (animal feed flour and corn flour) and decreased levels in fractions processed from inner portions, such as corn meal and flaking grits. The changes in concentration of aflatoxin M1 during manufacture of skim milk powder were assessed by Deveci & Sezgin (2006) in cow's milk contaminated artificially with aflatoxin M1 (AFM1) at two different levels. Pasteurization, concentration, and spray drying reduced the AFM1 contents with efficiencies dependant on the contamination levels. Park & Kim (2006) studied the effect of pressure cooking on aflatoxin residues in polished rice. They found aflatoxin losses of 78-88% after pressure cooking, which correlated with the reduction of aflatoxin-induced mutagenic potential. Interestingly, it was recently showed that raw material contamination with aflatoxins substantially affected the course of subsequent fermentation phases of maize mashes, influencing on characteristic factors such as alcohol concentration, productivity, yield and energy (Klosowski et al., 2010), and altering the composition of alcoholic fermentation volatile by-products in raw spirits (Klosowski & Mikulski, 2010). Notwithstanding the development of new technological strategies tending to reduce the food contamination with aflatoxins; the possible formation of mycotoxin degradation products (even more toxic that the mycotoxin itself), during the application of such technologies should be considered in all cases. Most of the food processes have variable effects on mycotoxins, with those that utilize the highest temperatures having greatest effects. Physical, chemical and biological methods of decontamination of aflatoxin-contaminated materials are another strategy feasible of application to avoid the toxicological effects of aflatoxins in human beings and in animals. Frequently, the physical reduction of the mycotoxin contents is achieved by the mechanical removal of highly contaminated fractions. Aly & Hathout (2011) suggested that the manufacture of hydrolyzed vegetable protein is a suitable method for decontamination of aflatoxin in highly contaminated grains, especially gluten fractions. This was concluded by the application of hydrochloric acid to aflatoxin B1 contaminated corn gluten. Enzymes of diverse origins may also be a useful tool to reduce the aflatoxin contamination of different matrices, as it was suggested by Wang et al. (2011). In line with these observations, microorganisms may have advantages with respect to the use of a purified enzyme, since they provide a subset of metabolic pathways that potentially could reduce the aflatoxins levels (Guan et al., 2010). 3.5 Approaches in the articles regarding to the toxicology of aflatoxins The determination of quantitative or qualitative value of risk related to the exposure of population to aflatoxins is a major concern, and is currently under evaluation in different geographic areas. Sugita-Konishi and co-workers (2010) evaluated the risk of exposure to aflatoxin B1 in Japan on the basis of the contamination of 24 foods from a 3-year retail market survey and data available on the food consumption by the population studied.

The Evolutionary Dynamics in the Research on Aflatoxins During the 2001-2010 Decade

315

Probabilistic approaches were used to estimate the aflatoxin B1 intake and the potential risk of cancer. The authors concluded that the current dietary intake of aflatoxin B1 in Japan has no appreciable effect on health. The main etiology of hepatocellular carcinoma (HCC) is chronic infection with hepatitis B and hepatitis C viruses. However, other important factors such as obesity, diabetes, nonalcoholic steatohepatitis and dietary exposures to food toxins like aflatoxins, were linked with the development of this disease. Emerging evidence suggests that the etiology of many cases of HCC is in fact multifactorial. A clear relationship between the exposure to high levels of aflatoxins and the prevalence of HCC was identified in several geographic areas; however the precise aflatoxin-virus interaction in the induction of this disease remains unclear. The current knowledge concerning the participation of aflatoxins in the induction of HCC was recently reviewed by Sanyal et al. (2010) and Whittaker et al. (2010). Biomarkers are currently used in the aflatoxins exposure assessment. They are classified as biomarkers of exposure (generally the aflatoxins itself or any of its metabolites, that can be measured in the body or after excretion from the body), effect (all the quantifiable changes that persists in an individual, which indicates the exposure to aflatoxins and may indicate a resulting health effect), and susceptibility (includes all natural characteristics of an organism that make it more susceptible to the effects of aflatoxins exposure). Johnson et al. (2010) studied the levels of biomarkers of exposure (serum AFB1-lysine adduct and urinary AFM1) in order to assess the aflatoxins exposure in a community with elevated incidence of hepatocellular carcinoma. The results of this survey suggest that the incidence and level of AF exposure were less than those observed in a high-risk population. Theumer et al. (2010) studied the changes in some biomarkers of effect by the co-exposure of spleen mononuclear cells to a mixture of aflatoxin B1 and fumonisins, and concluded that a possible protective effect of the fumonisins-AFB1 mixtures may exist with regards to the genotoxicity induced individually by the toxins. It was suggested that the CYP3A5 polymorphism found in the population of The Gambia, wich is associated with increased levels of the mutagenic AFB1-exo-8,9-epoxide particularly in individuals with low CYP3A4, may modulate individual risk of HCC (Wojnowski et al., 2004), and thus could be potentially used as a biomarker of susceptibility to aflatoxins. Metabonomics and proteomics are promising approaches to elucidate the biochemical perturbations of metabolism caused by aflatoxins. An overview of biochemical consequences of AFB1 exposure and comprehensive insights into the metabolic aspects of AFB1-induced hepatotoxicity in rats was reported by Zhang et al. (2011), who studied the AFB1-induced metabonomic changes in multiple biological matrices (plasma, urine, and liver) of rats to understand the mammalian systems responses to aflatoxin B1 exposure. Li and co-workers (2008) performed a proteome analysis of aflatoxin B1-induced hepatocarcinogenesis in tree shrew (Tupaia belangeri chinensis) In order to explore the proteins responsible for HCC. They found a protein that differentially expressed in hepatocellular carcinoma (Peroxiredoxin II), with a probable important role in hepatocarcinogenesis, possibly through its function in regulating peroxidation and hereby providing a favorable microenvironment for cancer cell surviving and progressing. Several studies were conducted in the 2001-2010 decade to characterize the impact of aflatoxins in the production performance of animals. For instance, Pandey & Chauhan (2007) found alteration of several productive parameters, including weight gain, feed intake, feed gain ratio, age at sexual maturity, production and quality of eggs and retention of nutrients; in White Leghorn female chicks exposed to different levels of aflatoxins.

316

Aflatoxins – Biochemistry and Molecular Biology

The toxicity of aflatoxins to the central nervous, digestive, renal, reproductive and immunological systems was evaluated in multiple experimental designs with microorganisms, cell lines and primary cells from laboratory animal exposed to aflatoxins. The immunotoxic effects of aflatoxins were studied by Theumer et al. (2003) in subchronic mycotoxicoses in Wistar rats poisoned with aflatoxin B1 alone and mixed with fumonisins. Essential oils from plants, a wide spectrum of chemical substances, and microorganisms were co-administered with the aflatoxins with the aim of identify possible protective effects against the toxicity of afatoxins. Sghaier et al. (2010) found antimutagenic effects of the essential oil extracted from the aerial parts of Teucrium ramosissimum against aflatoxin B1, in the bacterial reverse mutation assay in Salmonella typhimurium TA98, TA100, and TA1535. In a similar experimental system, Polivkova and co-workers (2010) observed antimutagenic effects of lycopene and tomato puree, wich was related with a significant reduction of DNA damage in mice cells assessed by the micronucleus test. Probiotics microorganisms could be used in order to amelliorate the adverse effects of exposure to aflatoxins (Kabak et al., 2009), since it was suggested that whole microbiological cells or fractions of them could adsorb mycotoxins, including the aflatoxins. 3.6 Approaches in the articles regarding to methodologies One of the leading advances in the 2001-2010 decade, concerning the analytical methodologies feasible of being applied in the field of aflatoxins, was the development of methods to quantify multiple mycotoxins including aflatoxins in several grains and byproducts. For instance, Zachariasova et al. (2010) developed and optimized a simple, high-throughput method for the control of 32 mycotoxins (Fusarium and Alternaria toxins, aflatoxins, ergot alkaloids, ochratoxins, and sterigmatocystin) in beer. Studies aimed to compare the performance of analytical methods were conducted by diverse laboratories. Zhang & Chen (2004) compared the post-column derivatization technique for the determination of aflatoxins B1, B2, G1 and G2 and the rapid procedure with fluorometric analysis for the determination of total aflatoxins, concluding that the later was not the suitable method for the determination of total aflatoxins in medicinal herbs and plant extracts. Rahamani et al. (2010) described the method validation for quantitative analysis of aflatoxins, ochratoxin A and zearalenone in cereals using HPLC with fluorescence detector; and concluded that it is suitable for the simultaneous determination of aflatoxins, ochratoxin A and zearalenone in cereals and is suitable for routine analysis. The potential pre-analytical sources of error in the quantification of aflatoxins in foods were also considered in the 2001-2010 decade. Gallo et al. (2010) highlighted the possibility of underestimating the food contamination with aflatoxins by inefficient extraction procedures in cattle feed containing adsorbents. Brera and co-workers (2010) emphasized the use of proper sampling methods throughout the agri-food chain when it comes to effectively detecting contaminants in foods and feeds.

4. Acknowledgment This work was partially supported by grants from Secretaría de Ciencia y Técnica de la Universidad Nacional de Córdoba (SeCyT-UNC), Agencia Nacional de Ciencia y Tecnología grants PICT 2005 Nº 15-32256 and PICT 2010 N° 1232, and Ministerio de Ciencia y Tecnología de la provincia de Córdoba grant 0279-005429/2006. Martin G. Theumer is career investigator of the National Research Council of Argentina (CONICET).

The Evolutionary Dynamics in the Research on Aflatoxins During the 2001-2010 Decade

317

5. References Aly, S.E. & Hathout, A.S. (2011). Fate of aflatoxin B(1) in contaminated corn gluten during acid hydrolysis. Journal of the Science of Food and Agriculture Vol. 91, No. 3, (Feb), pp. 421-427, ISSN 1097-0010 (Electronic), 0022-5142 (Linking) Aydin, A., Aksu, H. & Gunsen, U. (2010). Mycotoxin levels and incidence of mould in Turkish rice. Environ Monit Assess, (Sep 9), ISSN 1573-2959 (Electronic), 0167-6369 (Linking) Baker, R.L., Brown, R.L., Chen, Z.Y., Cleveland, T.E. & Fakhoury, A.M. (2009a). A maize lectin-like protein with antifungal activity against Aspergillus flavus. Journal of Food Protection Vol. 72, No. 1, (Jan), pp. 120-127, ISSN 0362-028X (Print), 0362-028X (Linking) Baker, R.L., Brown, R.L., Chen, Z.Y., Cleveland, T.E. & Fakhoury, A.M. (2009b). A maize trypsin inhibitor (ZmTIp) with limited activity against Aspergillus flavus. Journal of Food Protection Vol. 72, No. 1, (Jan), pp. 185-188, ISSN 0362-028X (Print), 0362-028X (Linking) Bankole, S.A. & Mabekoje, O.O. (2004). Occurrence of aflatoxins and fumonisins in preharvest maize from south-western Nigeria. Food Additives and Contaminants Vol. 21, No. 3, (Mar), pp. 251-255, ISSN 0265-203X (Print), 0265-203X (Linking) Brera, C., De Santis, B., Prantera, E., Debegnach, F., Pannunzi, E., Fasano, F., Berdini, C., Slate, A.B., Miraglia, M. & Whitaker, T.B. (2010). Effect of sample size in the evaluation of "in-field" sampling plans for aflatoxin B(1) determination in corn. Journal of Agricultural and Food Chemis ry Vol. 58, No. 15, (Aug 11), pp. 8481-8489, ISSN 1520-5118 (Electronic), 0021-8561 (Linking) Castells, M., Marin, S., Sanchis, V. & Ramos, A.J. (2008). Distribution of fumonisins and aflatoxins in corn fractions during industrial cornflake processing. International Journal of Food Microbiology Vol. 123, No. 1-2, (Mar 31), pp. 81-87, ISSN 0168-1605 (Print), 0168-1605 (Linking) Cleveland, T.E., Carter-Wientjes, C.H., De Lucca, A.J. & Boue, S.M. (2009). Effect of soybean volatile compounds on Aspergillus flavus growth and aflatoxin production. J Food Sci Vol. 74, No. 2, (Mar), pp. H83-87, ISSN 1750-3841 (Electronic), 0022-1147 (Linking) Chen, Z.Y., Brown, R.L., Rajasekaran, K., Damann, K.E. & Cleveland, T.E. (2006). Identification of a Maize Kernel Pathogenesis-Related Protein and Evidence for Its Involvement in Resistance to Aspergillus flavus Infection and Aflatoxin Production. Phytopathology Vol. 96, No. 1, (Jan), pp. 87-95, ISSN 0031-949X (Print), 0031-949X (Linking) Deveci, O. & Sezgin, E. (2006). Changes in concentration of aflatoxin M1 during manufacture and storage of skim milk powder. Journal of Food Protection Vol. 69, No. 3, (Mar), pp. 682-685, ISSN 0362-028X (Print), 0362-028X (Linking) Dorner, J.W. (2009). Biological control of aflatoxin contamination in corn using a nontoxigenic strain of Aspergillus flavus. Journal of Food Protection Vol. 72, No. 4, (Apr), pp. 801-804, ISSN 0362-028X (Print), 0362-028X (Linking) Dorner, J.W. & Horn, B.W. (2007). Separate and combined applications of nontoxigenic Aspergillus flavus and A. parasiticus for biocontrol of aflatoxin in peanuts.

318

Aflatoxins – Biochemistry and Molecular Biology

Mycopathologia Vol. 163, No. 4, (Apr), pp. 215-223, ISSN 0301-486X (Print), 0301486X (Linking) Gallo, A., Masoero, F., Bertuzzi, T., Piva, G. & Pietri, A. (2010). Effect of the inclusion of adsorbents on aflatoxin B1 quantification in animal feedstuffs. Food Addit Contam Part A Chem Anal Control Expo Risk Assess Vol. 27, No. 1, (Jan), pp. 54-63, ISSN 19440057 (Electronic), 1944-0057 (Linking) Gandomi, H., Misaghi, A., Akhondzadeh Basti, A., Hamedi, H. & Ramezani Shirvani, Z. (2010). Effect of Zataria multiflora Boiss. essential oil on colony morphology and ultrastructure of Aspergillus flavus. Mycoses, (Oct 11), ISSN 1439-0507 (Electronic), 0933-7407 (Linking) Gao, X., Brodhagen, M., Isakeit, T., Brown, S.H., Gobel, C., Betran, J., Feussner, I., Keller, N.P. & Kolomiets, M.V. (2009). Inactivation of the lipoxygenase ZmLOX3 increases susceptibility of maize to Aspergillus spp. Molecular Plant-Microbe Interactions Vol. 22, No. 2, (Feb), pp. 222-231, ISSN 0894-0282 (Print), 0894-0282 (Linking) Goncalez, E., Felicio, J.D. & Pinto, M.M. (2001). Biflavonoids inhibit the production of aflatoxin by Aspergillus flavus. Brazilian Journal of Medical and Biological Research Vol. 34, No. 11, (Nov), pp. 1453-1456, ISSN 0100-879X (Print), 0100-879X (Linking) Guan, S., Zhao, L., Ma, Q., Zhou, T., Wang, N., Hu, X. & Ji, C. (2010). In Vitro Efficacy of Myxococcus fulvus ANSM068 to Biotransform Aflatoxin B(1). Int J Mol Sci Vol. 11, No. 10, pp. 4063-4079, ISSN 1422-0067 (Electronic), 1422-0067 (Linking) Guo, B.Z., Xu, G., Cao, Y.G., Holbrook, C.C. & Lynch, R.E. (2006). Identification and characterization of phospholipase D and its association with drought susceptibilities in peanut (Arachis hypogaea). Planta Vol. 223, No. 3, (Feb), pp. 512520, ISSN 0032-0935 (Print), 0032-0935 (Linking) IARC, 1993. IARC Monographs on the evaluation of carcinogenic risks to humans., Some naturally occurring substances: Food items and constituents, heterocyclic aromatic amines and mycotoxins. Aflatoxins, International Agency for Research on Cancer, Lyon, pp. 249-395 Johnson, N.M., Qian, G., Xu, L., Tietze, D., Marroquin-Cardona, A., Robinson, A., Rodriguez, M., Kaufman, L., Cunningham, K., Wittmer, J., Guerra, F., Donnelly, K.C., Williams, J.H., Wang, J.S. & Phillips, T.D. (2010). Aflatoxin and PAH exposure biomarkers in a U.S. population with a high incidence of hepatocellular carcinoma. Science of the Total Environment Vol. 408, No. 23, (Nov 1), pp. 6027-6031, ISSN 18791026 (Electronic), 0048-9697 (Linking) Juglal, S., Govinden, R. & Odhav, B. (2002). Spice oils for the control of co-occurring mycotoxin-producing fungi. Journal of Food Protection Vol. 65, No. 4, (Apr), pp. 683687, ISSN 0362-028X (Print), 0362-028X (Linking) Kabak, B., Brandon, E.F., Var, I., Blokland, M. & Sips, A.J. (2009). Effects of probiotic bacteria on the bioaccessibility of aflatoxin B(1) and ochratoxin A using an in vitro digestion model under fed conditions. Journal of Environmental Science and Health. Part B: Pesticides, Food Contaminants, and Agricultural Wastes Vol. 44, No. 5, (Jun), pp. 472480, ISSN 1532-4109 (Electronic), 0360-1234 (Linking)

The Evolutionary Dynamics in the Research on Aflatoxins During the 2001-2010 Decade

319

Kensler, T.W., Roebuck, B.D., Wogan, G.N. & Groopman, J.D. (2011). Aflatoxin: a 50-year odyssey of mechanistic and translational toxicology. Toxicological Sciences Vol. 120 Suppl 1, (Mar), pp. S28-48, ISSN 1096-0929 (Electronic), 1096-0929 (Linking) Klosowski, G. & Mikulski, D. (2010). The effect of raw material contamination with mycotoxins on the composition of alcoholic fermentation volatile by-products in raw spirits. Bioresour Technol Vol. 101, No. 24, (Dec), pp. 9723-9727, ISSN 1873-2976 (Electronic), 0960-8524 (Linking) Klosowski, G., Mikulski, D., Grajewski, J. & Blajet-Kosicka, A. (2010). The influence of raw material contamination with mycotoxins on alcoholic fermentation indicators. Bioresour Technol Vol. 101, No. 9, (May), pp. 3147-3152, ISSN 1873-2976 (Electronic), 0960-8524 (Linking) Kong, Q., Shan, S., Liu, Q., Wang, X. & Yu, F. (2010). Biocontrol of Aspergillus flavus on peanut kernels by use of a strain of marine Bacillus megaterium. International Journal of Food Microbiology Vol. 139, No. 1-2, (Apr 30), pp. 31-35, ISSN 1879-3460 (Electronic), 0168-1605 (Linking) Kumar, R., Mishra, A.K., Dubey, N.K. & Tripathi, Y.B. (2007). Evaluation of Chenopodium ambrosioides oil as a potential source of antifungal, antiaflatoxigenic and antioxidant activity. International Journal of Food Microbiology Vol. 115, No. 2, (Apr 10), pp. 159-164, ISSN 0168-1605 (Print), 0168-1605 (Linking) Li, Y., Qin, X., Cui, J., Dai, Z., Kang, X., Yue, H., Zhang, Y., Su, J., Cao, J., Ou, C., Yang, C., Duan, X. & Liu, Y. (2008). Proteome analysis of aflatoxin B1-induced hepatocarcinogenesis in tree shrew (Tupaia belangeri chinensis) and functional identification of candidate protein peroxiredoxin II. Proteomics Vol. 8, No. 7, (Apr), pp. 1490-1501, ISSN 1615-9861 (Electronic), 1615-9853 (Linking) Mably, M., Mankotia, M., Cavlovic, P., Tam, J., Wong, L., Pantazopoulos, P., Calway, P. & Scott, P.M. (2005). Survey of aflatoxins in beer sold in Canada. Food Additives and Contaminants Vol. 22, No. 12, (Dec), pp. 1252-1257, ISSN 0265-203X (Print), 0265203X (Linking) Mohammadipour, M., Mousivand, M., Salehi Jouzani, G. & Abbasalizadeh, S. (2009). Molecular and biochemical characterization of Iranian surfactin-producing Bacillus subtilis isolates and evaluation of their biocontrol potential against Aspergillus flavus and Colletotrichum gloeosporioides. Canadian Journal of Microbiology Vol. 55, No. 4, (Apr), pp. 395-404, ISSN 0008-4166 (Print), 0008-4166 (Linking) Mwanda, O.W., Otieno, C.F. & Omonge, E. (2005). Acute aflatoxicosis: case report. East African Medical Journal Vol. 82, No. 6, (Jun), pp. 320-324, ISSN 0012-835X (Print), 0012-835X (Linking) Osman, N., El-Sabban, F.F., Al Khawli, A. & Mensah-Brown, E.P. (2004). Effect of foodstuff contamination by aflatoxin on the one-humped camel (Camelus dromedarius) in Al Ain, United Arab Emirates. Aust Vet J Vol. 82, No. 12, (Dec), pp. 759-761, ISSN 00050423 (Print), 0005-0423 (Linking) Palumbo, J.D., Baker, J.L. & Mahoney, N.E. (2006). Isolation of bacterial antagonists of Aspergillus flavus from almonds. Microbial Ecology Vol. 52, No. 1, (Jul), pp. 45-52, ISSN 0095-3628 (Print), 0095-3628 (Linking)

320

Aflatoxins – Biochemistry and Molecular Biology

Pandey, I. & Chauhan, S.S. (2007). Studies on production performance and toxin residues in tissues and eggs of layer chickens fed on diets with various concentrations of aflatoxin AFB1. British Poultry Science Vol. 48, No. 6, (Dec), pp. 713-723, ISSN 14661799 (Electronic), 0007-1668 (Linking) Park, D.L. (2002). Effect of processing on aflatoxin. Advances in Experimental Medicine and Biology Vol. 504, pp. 173-179, ISSN 0065-2598 (Print), 0065-2598 (Linking) Park, J.W. & Kim, Y.B. (2006). Effect of pressure cooking on aflatoxin B1 in rice. Journal of Agricultural and Food Chemis ry Vol. 54, No. 6, (Mar 22), pp. 2431-2435, ISSN 00218561 (Print), 0021-8561 (Linking) Passone, M.A., Resnik, S. & Etcheverry, M.G. (2007). Antiaflatoxigenic property of food grade antioxidants under different conditions of water activity in peanut grains. International Journal of Food Microbiology Vol. 118, No. 1, (Aug 15), pp. 8-14, ISSN 0168-1605 (Print), 0168-1605 (Linking) Penna, M.L. & Etcheverry, M. (2006). Impact on growth and aflatoxin B1 accumulation by Kluyveromyces isolates at different water activity conditions. Mycopathologia Vol. 162, No. 5, (Nov), pp. 347-353, ISSN 0301-486X (Print), 0301-486X (Linking) Pietri, A., Bertuzzi, T., Pallaroni, L. & Piva, G. (2004). Occurrence of mycotoxins and ergosterol in maize harvested over 5 years in Northern Italy. Food Additives and Contaminants Vol. 21, No. 5, (May), pp. 479-487, ISSN 0265-203X (Print), 0265-203X (Linking) Polivkova, Z., Smerak, P., Demova, H. & Houska, M. (2010). Antimutagenic effects of lycopene and tomato puree. J Med Food Vol. 13, No. 6, (Dec), pp. 1443-1450, ISSN 1557-7600 (Electronic), 1096-620X (Linking) Probst, C., Njapau, H. & Cotty, P.J. (2007). Outbreak of an acute aflatoxicosis in Kenya in 2004: identification of the causal agent. Applied and Environmental Microbiology Vol. 73, No. 8, (Apr), pp. 2762-2764, ISSN 0099-2240 (Print), 0099-2240 (Linking) Rahmani, A., Jinap, S. & Soleimany, F. (2010). Validation of the procedure for the simultaneous determination of aflatoxins ochratoxin A and zearalenone in cereals using HPLC-FLD. Food Addit Contam Part A Chem Anal Control Expo Risk Assess Vol. 27, No. 12, (Dec), pp. 1683-1693, ISSN 1944-0057 (Electronic), 1944-0057 (Linking) Reverberi, M., Fabbri, A.A., Zjalic, S., Ricelli, A., Punelli, F. & Fanelli, C. (2005). Antioxidant enzymes stimulation in Aspergillus parasiticus by Lentinula edodes inhibits aflatoxin production. Applied Microbiology and Biotechnology Vol. 69, No. 2, (Nov), pp. 207-215, ISSN 0175-7598 (Print), 0175-7598 (Linking) Rocha, L.O., Nakai, V.K., Braghini, R., Reis, T.A., Kobashigawa, E. & Correa, B. (2009). Mycoflora and co-occurrence of fumonisins and aflatoxins in freshly harvested corn in different regions of Brazil. Int J Mol Sci Vol. 10, No. 11, (Nov), pp. 5090-5103, ISSN 1422-0067 (Electronic), 1422-0067 (Linking) Sangare-Tigori, B., Moukha, S., Kouadio, H.J., Betbeder, A.M., Dano, D.S. & Creppy, E.E. (2006). Co-occurrence of aflatoxin B1, fumonisin B1, ochratoxin A and zearalenone in cereals and peanuts from Cote d'Ivoire. Food Additives and Contaminants Vol. 23, No. 10, (Oct), pp. 1000-1007, ISSN 0265-203X (Print), 0265-203X (Linking)

The Evolutionary Dynamics in the Research on Aflatoxins During the 2001-2010 Decade

321

Sanyal, A.J., Yoon, S.K. & Lencioni, R. (2010). The etiology of hepatocellular carcinoma and consequences for treatment. The Oncologist Vol. 15 Suppl 4, pp. 14-22, ISSN 1549490X (Electronic), 1083-7159 (Linking) Sghaier, M.B., Boubaker, J., Neffati, A., Limem, I., Skandrani, I., Bhouri, W., Bouhlel, I., Kilani, S., Chekir-Ghedira, L. & Ghedira, K. (2010). Antimutagenic and antioxidant potentials of Teucrium ramosissimum essential oil. Chem Biodivers Vol. 7, No. 7, (Jul), pp. 1754-1763, ISSN 1612-1880 (Electronic), 1612-1872 (Linking) Stenske, K.A., Smith, J.R., Newman, S.J., Newman, L.B. & Kirk, C.A. (2006). Aflatoxicosis in dogs and dealing with suspected contaminated commercial foods. J Am Vet Med Assoc Vol. 228, No. 11, (Jun 1), pp. 1686-1691, ISSN 0003-1488 (Print), 0003-1488 (Linking) Sugita-Konishi, Y., Sato, T., Saito, S., Nakajima, M., Tabata, S., Tanaka, T., Norizuki, H., Itoh, Y., Kai, S., Sugiyama, K., Kamata, Y., Yoshiike, N. & Kumagai, S. (2010). Exposure to aflatoxins in Japan: risk assessment for aflatoxin B1. Food Addit Contam Part A Chem Anal Control Expo Risk Assess Vol. 27, No. 3, (Mar), pp. 365-372, ISSN 19440057 (Electronic), 1944-0057 (Linking) Theumer, M.G., Canepa, M.C., Lopez, A.G., Mary, V.S., Dambolena, J.S. & Rubinstein, H.R. (2010). Subchronic mycotoxicoses in Wistar rats: assessment of the in vivo and in vitro genotoxicity induced by fumonisins and aflatoxin B(1), and oxidative stress biomarkers status. Toxicology Vol. 268, No. 1-2, (Jan 31), pp. 104-110, ISSN 1879-3185 (Electronic), 0300-483X (Linking) Theumer, M.G., Lopez, A.G., Masih, D.T., Chulze, S.N. & Rubinstein, H.R. (2003). Immunobiological effects of AFB1 and AFB1-FB1 mixture in experimental subchronic mycotoxicoses in rats. Toxicology Vol. 186, No. 1-2, (Apr 15), pp. 159-170, ISSN 0300-483X (Print), 0300-483X (Linking) Wang, J., Ogata, M., Hirai, H. & Kawagishi, H. (2011). Detoxification of aflatoxin B1 by manganese peroxidase from the white-rot fungus Phanerochaete sordida YK-624. FEMS Microbiology Letters Vol. 314, No. 2, (Jan), pp. 164-169, ISSN 1574-6968 (Electronic), 0378-1097 (Linking) Whittaker, S., Marais, R. & Zhu, A.X. (2010). The role of signaling pathways in the development and treatment of hepatocellular carcinoma. Oncogene Vol. 29, No. 36, (Sep 9), pp. 4989-5005, ISSN 1476-5594 (Electronic), 0950-9232 (Linking) Wojnowski, L., Turner, P.C., Pedersen, B., Hustert, E., Brockmoller, J., Mendy, M., Whittle, H.C., Kirk, G. & Wild, C.P. (2004). Increased levels of aflatoxin-albumin adducts are associated with CYP3A5 polymorphisms in The Gambia, West Africa. Pharmacogenetics Vol. 14, No. 10, (Oct), pp. 691-700, ISSN 0960-314X (Print), 0960314X (Linking) Xu, J., Wang, H., Ji, R. & Luo, X. (2003). [Study on the effect of the growth and aflatoxin production by Aspergillus flavus parasiticus NRRL 2999 in the present of Lactobacillus plantarum ATCC 8014]. Wei Sheng Yan Jiu Vol. 32, No. 4, (Jul), pp. 334-338, ISSN 1000-8020 (Print), 1000-8020 (Linking) Zachariasova, M., Cajka, T., Godula, M., Malachova, A., Veprikova, Z. & Hajslova, J. (2010). Analysis of multiple mycotoxins in beer employing (ultra)-high-resolution mass

322

Aflatoxins – Biochemistry and Molecular Biology

spectrometry. Rapid Communications in Mass Spectrometry Vol. 24, No. 22, (Nov 30), pp. 3357-3367, ISSN 1097-0231 (Electronic), 0951-4198 (Linking) Zhang, L., Ye, Y., An, Y., Tian, Y., Wang, Y. & Tang, H. (2011). Systems responses of rats to aflatoxin B1 exposure revealed with metabonomic changes in multiple biological matrices. J Proteome Res Vol. 10, No. 2, (Feb 4), pp. 614-623, ISSN 1535-3907 (Electronic), 1535-3893 (Linking) Zhang, X.H. & Chen, J.M. (2004). [Comparison between the post-column derivatization with bromine by HPLC and the fluorometric analysis for determination of aflatoxins in medicinal herbs and plant extracts]. Yao Xue Xue Bao Vol. 39, No. 12, (Dec), pp. 9971000, ISSN 0513-4870 (Print), 0513-4870 (Linking)

16 Binding of Aflatoxin B1 to Lactic Acid Bacteria and Saccharomyces cerevisiae in vitro: A Useful Model to Determine the Most Efficient Microorganism Romina P. Pizzolitto, Dante J. Bueno, María R. Armando, Lilia Cavaglieri, Ana M. Dalcero and Mario A. Salvano Universidad Nacional de Río Cuarto Argentina 1. Introduction Mycotoxins are toxic fungal metabolites found as contaminants in many agricultural products. Feeds contaminated with mycotoxins have a health risk to animals and, as a consequence, may cause big economical losses due to the low efficacy of animal husbandry (Richard, 2007). In addition, directly or indirectly (animal by-products) contaminated foods may also have a health risk to humans (CAST, 2003; Hussein & Brasel, 2001; Wild, 2007). Aflatoxins (AFs), a group of potent mycotoxins with mutagnic, carcinogenic, teratogenic, hepatotoxic and immunosupresive properties, are of particular importance because of their major occurrence and adverse effects on animal and human health, generalized as “aflatoxicosis” (CAST, 2003; Hussein & Brasel, 2001; Magnoli et al., 2011). The AFs are produced by genus Aspergillus, mainly A. flavus, A. parasiticus and A. nomius, that grow on a variety of raw material during growth, harvest, storage and transportation of for example, the cereal used in the preparation of food and feed commodities (Ito et al., 2001; Kurtzman et al., 1987; Payne, 1998; Pereyra et al., 2010). The investigation of strategies to prevent the presence of AFs in foods, as well as, to eliminate, inactivate or reduce the bio-availability of these mycotoxins in contaminated products include physical, chemical, and biological methods (Bueno et al., 2001; CAST, 2003; Kabak et al., 2006). Limitations such as the loss of nutritional and sensory qualities of the product, the expensive equipment required for these techniques and the impossibility to guarantee the desired results, have allowed us to consider the hipothesis that foods and feeds can always be potentially contaminated with aflatoxins. For instance, in the poultry industry aflatoxin B1 (AFB1) is almost an unavoidable feed contaminant and levels from 0200 ng/g have been reported (Dalcero et al., 1997). On the other hand, it is known that lactic acid bacteria (LAB) and some yeast, principally Saccharomyces cerevisiae, are capable to bind AFs in liquid media, apparently to cell wall components, polysaccharides and peptidoglycans of LAB (Haskard et al., 2001; Latinen et al., 2004) and glucomannans of yeast (Karaman et al., 2005; Raju & Devegowda, 2000) and

324

Aflatoxins – Biochemistry and Molecular Biology

therefore could be used as potential mycotoxin decontaminating (Armando et al., 2011; ElNezami et al., 1998; Haskard et al., 2000, 2001; Hernandez-Mendoza et al., 2009; Lee et al., 2003; Peltonen et al., 2001; Shetty et al., 2007). The inclusion of appropriate microorganisms in the contaminated diet could prevent the absorption of mycotoxins during their passage in the gastrointestinal tract and eliminated in the faeces (Bueno et al., 2007; El-Nezami et al., 2000; Gratz et al., 2004, 2007). Moreover, Kankaanpää et al. (2000) showed that the binding of AFB1 to the surface of LAB reduced their adhesive properties, and the accumulation of aflatoxins in the intestine may therefore be reduced via the increased excretion of an aflatoxin-bacteria complex. These considerations encouraged the recent emphasis on biological methods, but mainly focused on preventing AFs absorption in the gastrointestinal tract of the consumers, including these microorganisms in the diet and so prevent the aflatoxicosis effects. The first step in this direction is the selection of the most efficient microorganism for AFB1 removing and while many researchers have assayed LAB and yeast with AFB1 binding abilities (Ciegler et al., 1966; El-Nezami et al., 1998; Gourama & Bullerman, 1995; Haskard et al., 2001; Line et al., 1994; Oatley et al., 2000) no clear mechanism for this effect has been provided. Thus, this selection frequently is performed using a single concentration of AFB1, but we demonstrated that the microorganism efficiency may change when the mycotoxin concentration is modified (Bueno et al., 2007; Pizzolitto, 2011), therefore the microorganism selected could not be the most competent. In this context, we investigated the nature of the interaction between different microorganisms and AFB1 molecule, which led us to develop a model to explain the binding of AFB1 by LAB and Saccharomyces cerevisiae strains. This model allows an estimation of two important parameters related to a microorganism's capacity for dietary decontamination: the number of binding sites for AFB1 in the surface microorganism (M) and the equilibrium constant of the process involved (Keq), both of them are useful in the selection of the most suitable microorganism in a wide range of AFB1 concentration (Bueno et al., 2007). In adittion, studies of viability of the microorganisms in the salivary and gastrointestinal tract, cell adhesion, autoaggregation, coaggregation and antimicrobial activity against pathogen strains, were also evaluated as a way to research potential beneficial properties on the host (Armando et al., 2011). Thus, in this chapter we describe the development and application of an in vitro methodology to evaluate the aflatoxin B1 binding ability, gastrointestinal tolerance and potential beneficial properties of Saccharomyces cerevisiae strains, useful to select the more appropriated microorganism to be assayed in expensive, complicated but necessary in vivo studies. 2.1 Study of microorganism-aflatoxin B1 interaction To select accurately the most efficient microorganism to bind AFB1, is very important so as to protect against aflatoxicosis by inclusion of microorganisms in the diet. Usually, the methodology assayed has been a selection of several candidates using a unique mycotoxin concentration (Haskard et al., 2001; Hernandez-Mendoza et al., 2009; Peltonen et al., 2001; Shetty & Jespersen, 2006). Table 1, developed in our laboratory, is a clear example of this methodology and its analysis shows that the efficiency of the microorganisms is strain dependent, so that toxin removal ranged from 13 to 42% for LAB strains and 16 to 40% for the yeast strains tested.

Binding of Aflatoxin B1 to Lactic Acid Bacteria and Saccharomyces cerevisiae in vitro: A Useful Model to Determine the Most Efficient Microorganism

325

Microorganism

Source

% AFB1 bound ± SDa

Lactobacillus acidophilus Po22

Poultry cecum

42.8 ± 1.7

L. acidophilus Po7

Poultry

34.6 ± 1.6

L. acidophilus 24

Dairy

32.6 ± 2.0

L. casei 1

Dairy

27.6 ± 1.5

L. fermentum 23

Human

34.6 ± 3.2

L. acidophilus CRL 1014

ATCC collectionb

25.4 ± 1.7

L. fermentum subsp. cellobiosus 408

Poultry

13.2 ± 9.8

Saccharomyces cerevisiae RC016

Pig gut

15.8 ± 3.6

S. cerevisiae RC012

Feedstuff

29.6 ± 2.4

S. cerevisiae RC008

Feedstuff

20.6 ± 2.6

S. cerevisiae RC009

Feedstuff

16.4 ± 1.2

S. cerevisiae 01

Poultry faeces

28.6 ± 3.5

S. cerevisiae 03

Poultry faeces

26.6 ± 2.9

S. cerevisiae 05

Poultry faeces

33.4 ± 1.9

S. cerevisiae 08

Poultry faeces

36.4 ± 2.7

S. cerevisiae CECT 1891

STCC

collectionc

40.0 ± 2.5

a The percentage of AFB1 bound to cells was calculated as the difference between the total AFB1 (5 µg ml-1) and the amount of free AFB1 (supernatant). Values are means ± standard deviations for duplicate samples. bATCC, American Type Culture Collection, Manassas, VA, USA. c STCC, Spanish Type Culture Collection, University of Valencia, Valencia, Spain.

Table 1. Percentage of AFB1 bound to cells upon exposure to viable microorganisms Bacteria, 2 x 108 CFU ml-1 and yeast 1 x 107 CFU ml-1 were incubated with 1 ml of AFB1 (5 µg ml-1) in PBS for 30 min at 37°C. The microorganisms were then pelleted by centrifugation, and the supernatant was collected for free AFB1 analysis by HPLC according to Bueno et al. (2007). The same experiment, but using three different concentrations of aflatoxin B1, shows that the microorganism assayed to one concentration could not be the most efficient when the latter is changed (Table 2 and 3). Thus when AFB1 concentration was 50 ng ml-1, S. cerevisiae RC 016 was the most effective strain, but S. cerevisiae 08 and S. cerevisiae CECT 1891 were the best when AFB1 concentration was increased at 100 ng ml-1, and with 500 ng ml-1 of AFB1 S. cerevisiae RC 016 was again the most efficient strain. In addition, S. cerevisiae 01 and S. cerevisiae 03 removed AFB1 with similar ability when their concentrations were 50 and 100 ng ml-1; however S. cerevisiae 01 was more effective at 500 ng ml-1 (Table 2). Lactic acid bacteria strains also showed the same behaviour, because L. rhamnosus I, L. acidophilus 24 and L. casei subsp. rhamnosus were the best at 50, 100 and 500 ng ml-1 respectively (Table 3).

326

Aflatoxins – Biochemistry and Molecular Biology

AFB1 concentration (ng ml-1) 50

Strains

100

500

AFB1 binding (ng

ml-1)

%

(ng ml-1)

%

(ng ml-1)

%

S. cerevisiae CECT 1891

10.0 ± 4.3

20.0

57.6 ± 8.6

57.6

255.0 ± 32.1 51.0

S. cerevisiae RC 008

33.8 ± 0.1

67.6

45.6 ± 7.1

45.6

197.9 ± 24.1 38.2

S. cerevisiae RC 012

15.3 ± 1.6

29.6

21.5 ± 3.1

21.5

103.7 ± 9.4

20.2

S. cerevisiae RC 009

8.4 ± 0.8

16.8

21.5 ± 0.8

21.5

159.3 ± 1.2

31.8

S. cerevisiae RC 016

41.6 ± 1.9

82.0

49.1 ± 1.4

49.1

328.8 ± 5.2

65.7

S. cerevisiae 01

19.3 ± 1.2

38.6

31.7 ± 1.2

31.7

164.0 ± 6.9

32.8

S. cerevisiae 03

23.3 ± 2.9

46.6

34.5 ± 1.8

34.5

128.7 ± 5.8

25.7

S. cerevisiae 05

16.7 ± 1.2

33.4

24.0 ± 1.7

24.0

92.0 ± 6.9

18.4

S. cerevisiae 08

23.2 ± 2.8

46.4

58.9 ± 2.1

58.9

187.0 ± 18.2 37.4

Cells (107 CFU ml-1) were suspended in PBS in the presence of AFB1 at the indicated concentration and incubated as described in Table 1. AFB1 analysis by HPLC was performed according to Trucksess et al. (1994). Data are means ± standard deviations from three experiments in duplicate.

Table 2. Aflatoxin B1 remotion by Saccharomyces cerevisiae strains at three different mycotoxin concentrations AFB1 concentration (ng ml-1) 50

Strains

100

500

AFB1 binding (ng ml-1)

%

(ng ml-1)

%

(ng ml-1)

%

Lactobacillus casei subsp. rhamnosus

18.0 ± 3.4

36.0

56.3 ± 5.4

56.3

338.0 ± 7.2

67.6

L. rhamnosus I

35.8 ± 1.3

71.6

61.8 ± 3.1

61.8

254.7 ± 4.2

50.9

L. fermentum 23

22.3 ± 1.2

44.6

41.0 ± 2.8

41.0

225.3 ± 10.8

45.1

L. acidophilus 24

26.3 ± 1.9

52.6

82.5 ± 3.2

82.5

254.0 ± 25.9

50.8

L. casei 1

13.8 ± 0.9

27.6

27.5 ± 1.3

27.5

59.0 ± 1.7

11.8

Cells (5 x 108 CFU ml-1). AFB1 binding to cells was performed as described in Table 2. Data are means ± standard deviations from three experiments in duplicate.

Table 3. Aflatoxin B1 remotion by lactic acid bacteria strains at three different mycotoxin concentrations Therefore, as AFB1 concentration is highly variable in foods and feeds, the methods of selection using a unique aflatoxin B1 concentration may lead to erroneous results. Another

Binding of Aflatoxin B1 to Lactic Acid Bacteria and Saccharomyces cerevisiae in vitro: A Useful Model to Determine the Most Efficient Microorganism

327

very important condition to consider is how to quantify the concentration of employed microorganisms, CFU ml-1 or total cells ml-1, where to evaluate whether viable and nonviable cells remove AFB1 with the same efficiency becomes necessary. Thus, the solution to these problems will be to know the mechanism involved in cellaflatoxin interaction, and probably will also allow us to find the microorganism able to protect against aflatoxicosis in a wide range of mycotoxin concentration. In this sense, one of the objectives of the present study was to develop a theoretical model able to explain the binding of AFB1 by LAB and Saccharomyces cerevisiae strains. With this purpose we studied as influences on the process involved the following conditions: microorganism-AFB1 time contact, aflatoxin B1 and microorganism concentration, cell viability, release of AFB1 bound by cells and importance of the microorganism cell wall. 2.1.1 Microorganism-AFB1 time contact Table 4 summarizes some representative results (three LAB and three yeast strains) of the studies done in our laboratory when contact time between AFB1 and the microorganisms were tested. By varying the incubation time, no significant difference in the amount of AFB1 removed for LAB and yeast strains were observed. Furthermore, the process was fast, since in 1 minute the microorganism was able to bind the same amount of mycotoxin as in 6 h. AFB1 Binding (ng ml-1) Time (min)

S. cerevisiae S. cerevisiae S. cerevisiae 08 RC016 CECT 1891

L. fermentum L. acidophilus subsp. L. casei 1 P22 cellobiosus 408

1

182.8 ± 18.2

318.5 ± 5.2

258.0 ± 14.2

61.5 ± 4.3

57.5 ± 2.2

89.4 ± 4.2

5

194.0 ± 12.6

312.8 ± 6.7

245.5 ± 10.3

64.3 ± 5.1

54.3 ± 3.1

91.8 ± 3.3

60

178.9 ± 15.7 332.0 ± 10.2

252.7 ± 15.6

62.8 ± 4.6

59.0 ± 4.2

88.9 ± 5.1

360

193.2 ± 14.6 326.6 ± 12.1

267.2 ± 12.6

63.8 ± 3.5

56.8 ± 2.9

92.3 ± 4.7

Table 4. Effect of incubation time on aflatoxin B1 binding by viable cells of yeasts and LAB AFB1 concentration: 0.5 µg ml-1. Cells number: yeasts 1 x 107 CFU ml-1; LAB 5 x 108 CFU ml-1. AFB1 binding was performed as described in Table 1, except that the incubation time varied as indicated in column 1. Data are means ± standard deviations from three experiments in duplicate. There is not significant differences (P AFG1 > AFB2 > AFG2, reflecting the role played by epoxidation of the 8,9-double bond and also the greater potency associated with the cyclopentenone ring of the B series, when compared with the six-membered lactone ring of the G series. AFM1 and AFM2 are hydroxylated forms of AFB1 and AFB2 (Mclean & Dutton, 1995; Wogan, 1966). In the primary fungi metabolism a lot of interrelated reactions catalyzed by enzymes occur, with the objective of promoting energy and primary metabolites (synthetic intermediates and macromolecules), ensuring the growth and reproduction of fungi. Secondary * Sergiane Souza Caldas2, Vivian Feddern3, Renata Heidtmann Bemvenuti2, Helen Cristina dos Santos Hackbart2, Michele Moraes de Souza2, Melissa dos Santos Oliveira4, Jaqueline Garda-Buffon2, Ednei Gilberto Primel2 and Eliana Badiale-Furlong2 1 Instituto Federal Catarinense – Campus Concórdia (IFC), Brazil 2 Federal University of Rio Grande (FURG), Brazil 3 Embrapa Swine and Poultry, Brazil 4 Instituto Federal Farroupilha – Campus Santo Augusto (IFF), Brazil

416

Aflatoxins – Biochemistry and Molecular Biology

metabolites are synthesized by a variety of routes from primary metabolites (Obrian et al., 2003; Ueno, 1986; Wild & Montesano, 2009). The biosynthesis of aflatoxins, as all secondary metabolites, is strongly dependent on growth conditions such as substrate composition or physical factors such as pH, water activity, temperature or modified atmospheres. Depending on the particular combination of external growth parameters the biosynthesis of aflatoxin can either be completely inhibited, albeit normal growth is still possible or the biosynthesis pathway can be fully activated. Knowledge about these relationships enables an assessment of which parameter combinations can control aflatoxin biosynthesis. The biochemical correlation between aflatoxin production and oxidative stress suggest that the latter is a prerequisite for aflatoxin synthesis (Ellis et al., 1993; Giorni et al., 2008; Luchese & Harrigan, 1993; Molina & Giannuzzi, 2002; Ribeiro et al., 2006; Schmidt-Heydt et al., 2009). The chapter gives a section on aflatoxin analysis, its occurrence in food and feed as well as its control, once aflatoxin is the major mycotoxin studied and thus is of great concern for human and animal’s health due to its carcinogenic, mutagenic, teratogenic and immunosupressive effects.

2. Factors promoting contamination in aflatoxins and occurrence Fungi which grow and produce toxins in grains during storage are influenced by factors related to inadequate moisture and temperature, combined with long residence time in warehouses, which are stressful situations and originate toxigenic potential outbreak (Dilkin, 2002). The most important factors that help predict the occurrence of aflatoxins in food include weather conditions (temperature and atmospheric humidity), agronomical practices (crop rotation and soil cultivation) and internal factors of the food chain (drying and storage conditions). A comprehensive approach is needed to identify and control risks related to food production system that could present a potential hazard to human health, being necessary to identify emerging risks which may include "newly" identified risks, not previously observed risks in human or animal food chain as well as known risks. The emerging risks need to be identified as early as possible in order to take appropriate preventive measures. Thus, the specific risk can be prevented from becoming a danger (Van der Fels-Klers et al., 2008). Several groups of researchers from the European Union reached a consensus on the most important indicators, based on three stages in food production chain. For cultivation stage the selected indicators were: relative humidity, temperature, crop rotation, tillage practices and water activity of seeds. For transportation and storage the following factors were included: water activity, relative humidity, ventilation, temperature, storage capacity and logistics. For processing the indicators were: data quality, the fraction of grain used, the water activity of seeds, implanted traceability and system quality (Park & Bos, 2007; Van der Fels-Klers et al., 2010). According to Park & Bos (2007) and Marvin et al. (2009a) to anticipate emerging risks models are developed to assess the risk from the indicators identified. The next step is to identify the sources of information for these indicators, such as climate change (changes in temperature and rainfall), market and consumer trends (crop demand, price and production) and market research (economics, as inflation and taxes) global trade (import and export data and trade barriers), transportation (strikes and transport company records), technology (covers of scientific journals), prevalence of pests, changes in legislation (registration of pesticides). The risk categories within each of the selected indicators should be defined for each specific food.

Aflatoxins: Contamination, Analysis and Control

417

Among the models currently available in the literature to predict the occurrence of fungi and mycotoxins, are the meteorological indicators in combination with agricultural information. With respect to management strategies, monitoring and prevention are the main indicators derived from the food chain (Dekkers et al., 2008). It is important to stand out the potential interactions among indicators which should be taken into account, for example: between relative humidity and temperature during cultivation; among storage conditions and drying and finally, between crop rotation and management policies (Van der Fels-Klers, 2010; Marvin et al., 2009b). Due to the great health concern in relation to mycotoxin contaminated food ingestion, studies are being conducted worldwide to verify the occurrence of aflatoxins. The main food products susceptible to fungal growth and consequently to mycotoxins’ production, include peanuts (raw, roasted, sweet and infrosted), corn (popcorn, hominy and grains), wheat, rice, nut, walnuts, hazelnuts, cashews, almonds, dried fruits, spices, cotton seed, cassava, vegetable oils, cocoa and others that are normally used in the composition of foods and feeds. Thus, animals are also subjected to aflatoxin contamination, and when meat and milk from these animals are ingested, human contamination may also occur (Kwiatkowski & Alves, 2007). Mycotoxins importance relies on harm caused to human and animal health, besides economical losses in agriculture (Amaral et al., 2006). Rubert et al. (2010) evaluated a total of 22 samples obtained from a local supermarket (10 samples of malt, 7 samples of coffee and 5 samples of instant-based cereal-breakfast beverage). Four samples of the total malt samples were positive for AFG2 and AFG1, and traces of AFB1 and AFB2 were detected. Khayoon et al. (2010) verified the occurrence of AFB1, B2, G1 and G2 in 42 animal feeds, comprising corn (16), soybean meal (8), mixed meal (13), sunflower, wheat, canola, palm kernel, copra meals (1 each). The results showed that eight samples (19%) were contaminated with aflatoxins, ranging from 6.5 to 101.9 ng g−1. Ibáñez-Vea et al. (2011) evaluated AFG2, AFG1 and ZEA mycotoxins in 20 barley samples. All of the samples analyzed presented levels of AFB1 above its LOD, but only 5 (25%) presented quantifiable levels (>LOQ), with 0.173 μg kg−1 and 0.185 μg kg−1 being the mean of the positive values and the maximum level found, respectively. Reiter et al. (2010) evaluated eighty-one rice samples purchased from different markets. The results revealed that AFB1 (0.45 to 9.86 μg kg−1) could be quantified in 15 samples and AFB2 (1.5 μg kg−1) in one sample. Matumba et al. (2010) investigated aflatoxins in sorghum grain and malt samples, traditional opaque sweet beverage (thobwa) and beer prepared from sorghum malts. All malt and beer samples, 15% and 43% of the sorghum and thobwa samples, respectively, were contaminated. The sorghum malt prepared for beer brewing, had a significantly (p < 0.01) total aflatoxin content (average 408 ± 68 μg kg-1) than any other type of sample. Dors et al. (2011) conducted a survey of mycotoxins in parboiled and whole rice. From the samples analyzed, 9% were contaminated with AFB1 in levels ranging from 11 to 74 g kg-1. Coelho et al. (1999) studied aflatoxin and ochratoxin A migration during rice parboiling process under different conditions of soaking, autoclaving and drying. It was noted that there was mycotoxin migration from the husk to the starchy endosperm in the following proportions: 32% AFB1, 44% AFB2, 36% AFG1 and 22% AFG2. Dors et al. (2009) assessed mycotoxin migration to the starchy endosperm during the parboiling process and the results showed a lower trend of migration from AFB1 in 6 h soaking and 30 min autoclaving. Amaral et al. (2006) examined 123 samples of food products based on corn and corn grain, of which 16 were positive with levels of 0.78 μg kg-1. Ramos et al. (2008) detected the presence

418

Aflatoxins – Biochemistry and Molecular Biology

of Aspergillus spp. and aflatoxin contamination grain samples (12) analyzed and this result was correlated with the greatest amount of rain during harvest. Levels of contamination ranged from "not detected" (nd) to 277.8 µg kg-1, for AFB1; from 0.7 to 14 µg kg-1 for AFB2; and from nd to 34.1 µg kg-1 for AFG2. Oliveira et al. (2010) found aflatoxin contamination in 70% of maize samples from criollo varieties, which have not undergone genetic intervention, at levels ranging from 1 to 2.6 µg kg-1. Almeida et al. (2009) collected 80 samples of maize for poultry feed in two feed mills, from these samples 10% were contaminated with levels varying from 1 to 5 mg kg-1. Marques (2007) analyzed 47 samples of corn grits for animal consumption and 46 were positive for aflatoxin with a maximum of 50 µg kg-1. D’Angelo et al. (2007) reported injury in calves for veal production that had a corn-based diet. The toxicological analysis of corn-based feed revealed contamination in the following levels: 1400 µg kg-1 AFB1, 120 µg kg-1 AFB2, 80 µg kg-1 AFG1 and 70 µg kg-1 AFG2. In the liver of three animals were found levels of total aflatoxins of 0.1, 0.3 and 0.6 µg kg-1. Velazquez et al. (2009) analyzed 40 samples of feed for dairy cattle and 92% of them were contaminated with aflatoxins at levels between 4.82 a 2.89 µg Kg-1. Most of AFB1 and AFB2 ingested by mammals is eliminated through urine and faeces, however a fraction is biotransformed in the liver and excreted together with milk in the form of aflatoxins AFM1 and AFM2, respectively. AFM1 could be detected in milk 12-24 h after the first AFB1 ingestion, reaching a high level after a few days. The ratio between AFB1 ingested and AFM1 excreted has been estimated to be 1-3%. One of the most used treatments for milk processing is heating, however, AFM1 is resistant to any thermal treatment (Carvajal et al., 2003; Park, 2002; Van Egmond, 1989). Rahimi et al. (2010) analyzed 311 samples of raw milk from cow, water buffalo, camel, sheep, and goat. AFM1 was found in 42.1% of the samples by average concentration of 43.3 ± 43.8 ng kg-1. The incidence rates of AFM1 in raw cow, water buffalo, camel, sheep, and goat milks were, 78.7%, 38.7%, 12.5%, 37.3%, and 27.1%, respectively. Fallah (2010) investigated the occurrence of AFM1 in 225 commercial liquid milk samples composed of pasteurized milk (116 samples) and UHT milk (109 samples). AFM1 was detected in 151 (67.1%) samples, consisted of 83 (71.5%) pasteurized milk samples (mean: 52.8 ng L-1 ; range: 5.8–528.5 ng L-1) and 68 (62.3%) UHT milk samples (mean: 46.4 ng L-1; range: 5.6– 515.9 ng L-1). Heshmati and Milani (2010) verified the levels of AFM1 in UHT milk samples. Two hundred and ten UHT milk samples were obtained from supermarkets in Tehran, Iran. AFM1 was found in 116 (55.2%) of 210 UHT milk samples examined. The levels of AFM1 in 70 (33.3%) samples were higher than the maximum tolerance limit (0.05 μg L-1) accepted by some European countries while none of the samples exceeded the prescribed limit of US regulations. The same authors also studied AFM1 levels of 61 milk samples delivered from small milking farms. The maximum mean concentrations of AFM1 recorded in winter– spring season were in the range of 35.8–58.6 ng L-1 and in summer–autumn season in the range of 11.6–14.9 ng L-1. Cano-Sancho et al. (2010) found AFM1 occurrence in the main dairy products consumed, that is 94.4% (68/72) of whole UHT milk samples, in 2.8% (2/72) of yoghurt samples, but was not detected in cheese. The maximum level was detected in one yoghurt sample with 51.58 ng kg-1. Martins & Martins (2004) determined the occurrence of AFM1 in 96 yoghurt samples, being 48 of them natural and 48 added by strawberry pieces. The results showed that 18.8% of the samples were contaminated with AFM1, being 2 samples of natural

Aflatoxins: Contamination, Analysis and Control

419

yoghurt (0.043 and 0.045 ug L-1) and 16 from fruit added yoghurt (0.019 and 0.098 ug L-1). Khoury et al. (2011) investigated the presence and levels of AFM1 in 138 dairy samples (milk and yogurt). Results obtained showed that AFM1 was found in 40.62 % and 32.81 % of milk and yogurt samples respectively. Fallah et al. (2009) studied 210 cheese samples composed of white cheese (116 samples) and cream cheese (94 samples). AFM1 at measurable level (50 ng kg-1) was detected in 161 (76.6%) samples, consisting of 93 (80.1%) white and 68 (72.3%) cream cheese samples. Dashti et al. (2009) evaluated a total of 321 milk samples (177 fresh, 105 long-life, 27 powdered milk and 12 human milk), 40 cheese samples and 84 feed samples were analyzed for AFM1. Results showed that all fresh milk samples except one were contaminated with AFM1 ranging from 4.9 to 68.7 ng kg-1, for the long-life milk samples were below the detection limit to (88.8 ng kg-1) while in powdered milk samples ranged from 2.04 to 4.14 ng kg-1. From human milk samples, only five were contaminated, with levels ranging from 8.83 to 15.2 ng kg-1. The cheese samples recorded 80% contamination with AFM1 with a range of 23.8–452 ng kg-1. Manetta et al. (2009) investigated samples of whey, curd and a typical hard and long maturing cheese such as Grana Padano produced with naturally contaminated milk in a range of 30–98 ng kg-1. Experimental results showed that, in comparison to milk, AFM1 concentration levels increased both in curd (3-fold) and in long maturing cheese (4.5fold), while AFM1 occurrence in whey decreased by 40%. Under review done by Montagna et al. (2008), there is an increase in aflatoxin M1 concentration as cheese ripening stage progresses, due to water loss and the consequent concentration of substances present. Sassahara et al. (2005) collected 98 feed and 42 raw milk samples and the results showed that there was contamination by AFM1 in 26% commercial feed samples, besides 53% of feed samples prepared at the farm and in 100% of corn samples used in animal nutrition. As a result of this aflatoxin incidence in animal diet, milk showed 24% contamination in the collected samples. Romero et al. (2010) evaluated the presence of AFM1 in human urine samples from a specific Brazilian population, as well as in corn, peanut, and milk consumption measured by two types of food inquiry. A total of 69 samples were analyzed and 45 of them (65%) presented contaminations 1.8 pg mL−1, which was the limit of quantification (LOQ). Seventy eight percent (n = 54) of the samples presented detectable concentrations of AFM1 (>0.6 pg mL−1). The AFM1 concentration among samples above LOQ ranged from 1.8 to 39.9 pg mL−1. There were differences in food consumption profile among donors, although no association was found between food consumption and AFM1 concentration in urine. The high frequency of positive samples suggests exposure to aflatoxins by the studied population. Aflatoxins are found in maize and peanuts, as well as in tree nuts and dried fruits (Zain, 2011). Nakai et al. (2008) evaluated the mycoflora and occurrence of aflatoxins in stored peanut samples (hulls and kernels). Analysis of hulls showed that 6.7% of the samples were contaminated with AFB1 and AFB2; in kernels, 33.3% of the samples were contaminated with AFB1 and 28.3% with AFB2. Analysis of the toxigenic potential revealed that 93.8% of the A. flavus strains isolated were producers of AFB1 and AFB2. Shenasi et al. (2002) detected aflatoxins in 12% of the samples at twenty-five varieties of dates (Phoenix dactylifera) although aflatoxigenic Aspergillus were detected in 40% of the varieties examined. Bircan (2009) tested aflatoxin contamination in 98 dried figs analyzed for OTA to determine the co-occurrence of both toxins. Seven samples were confirmed aflatoxin positive, in the range of 0.23–4.28 ng g-1 and only 2 samples contained both toxins, with a maximum concentration of 24.37 ng g-1 for OTA and 1.02 ng g-1 for AFB1.

420

Aflatoxins – Biochemistry and Molecular Biology

More recently, Herzallah et al. (2009) studied aflatoxin contamination in meat products collected in 5 different months. The AFB1, AFB2, AFG1 and AFG2 contents in the analysed food products ranged from 1.10 to 8.32 g.L-1 and 0.15 to 6.36 g.L-1 in imported and fresh meat samples collected during March, respectively. Fruits and vegetables do not appear to be of major concern as possible sources of mycotoxin contamination in food and feeds because they were only listed as minor sources in a statement of the Institute of Food Science and Technology Trust Fund (2006). Major sources on the list included mold damaged foodstuffs, specifically cereals and oilseeds. FAO has done a lot of work on mycotoxins in developing countries, although economic dimensions are rarely observed. In horticultural crops, mycotoxins are primarily associated with dried fruits (figs and prunes), certain processed products (apple and grape juice) and are probably in apples and grapes (Dombrink-Kurtzman, 2008). Although a large number of different mycotoxins exist, there are only a few of them that are regularly found in foods. Most reports concerning aflatoxin formation on fruits refer to figs or citrus fruits (Drusch & Ragab, 2003). Aflatoxins constitute a problem that is already present in the orchard. Little contamination occurs when firm, ripe fruits are dried immediately (Steiner et al., 1988). From a practical point of view, the best approach for eliminating mycotoxins from foods is to prevent mold growth at all levels of production, including harvesting, transport, and storage (Boutrif, 1998). Thus, the occurrence of fungi and mycotoxins can be controlled by applying a number of preventive measures both before and after harvest, including insect control, good harvesting, drying, and storage practices. If mycotoxin contamination has occurred, the levels of toxins can be reduced by physical, chemical or biological decontamination. Milling, food processing, and regulatory control of toxins to safety levels can also have a positive impact on food safety (Trucksess & Diaz-Amigo, 2011).

3. Sampling, measurement and analysis 3.1 Sample preparation Since AFs are inhomogeneous distributed in food and feed, high-contaminated hotspots can occur. Thus, sampling is an important step in the analysis of contaminated food and feed (Reiter et al., 2009). Relating to the sample preparation techniques used in the last years, liquid-solid extraction has been widely employed. Usually the procedure consists of weighing a mass of the homogenized sample, add the extractor solvent and agitate in a shaker. Commonly, after these steps, filtration is carried out. In these extractions different volumes and solvent kinds were employed. Solvent volumes ranging from 20 to 250 mL and composed mainly of methanol/water or acetonitrile/water have been used. The choice for the best extraction solvent is directly related to the extraction efficiency and the number of co-extractives that this solvent extracts. In the work developed by Capriotti et al. (2010) the authors compared the use of methanol, acetonitrile and acetone for mycotoxins’ extraction from cereals, being observed the highest recovery for the analytes in the acetone solution. Another tool that has been employed during extraction is the ultrasound assisted extraction (Amate et al., 2010; Bacaloni et al., 2008; Capriotti et al., 2010; Quinto et al., 2009). Ultrasound is a simple and versatile method because it aggressively agitates the solution system improving transfer from the cell into the solvent. Bacaloni et al. (2008) employed ultrasound extraction and compared the technique with matrix solid-phase dispersion (MSPD) and homogenization. Recoveries comparable to those obtained with the

Aflatoxins: Contamination, Analysis and Control

421

homogenization method were achieved with a sonication time of 10 min. The authors concluded that the employment of ultrasound is time-saving because it is easy to handle and many samples can be treated at the same time. Besides, ultrasonic extraction may be an efficient, safe and reliable alternative to homogenization and MSPD extractions. MSPD technique has been employed for aflatoxins’ extraction in food samples (Cavaliere et al., 2007; Rubert et al., 2010; Sebastià et al., 2010). MSPD involves the homogenization of the sample together with a suitable sorbent (usually octadecylsilica) using a pestle and mortar. The solid mixture is transferred to a cartridge and after, the aflatoxins are eluted and determined. Rubert et al. (2010) extracted the aflatoxins AFB1, AFB2, AFG1 and AFG2 from cereal using 1 g sample, 1 g C18 and 10 mL acetonitrile for the elution from the cartridges. Recoveries were reported to be between 64 and 91%, and limits of quantification of 1 µg kg-1 were reached. Pressurized fluid extraction (PFE), with trade name of accelerated solvent extraction (ASE) has also been employed for aflatoxins’ extraction (Sheibani & Ghaziaskar, 2009; Desmarchelier et al., 2010). This technique employs solvents at elevated pressures and temperatures to achieve complete extraction of analytes from solid and semi-solid samples with lower solvent volumes and shorter extraction times (Sheibani & Ghaziaskar, 2009). The accelerated extraction solvent was compared to QuEChERS procedure (acronym name for Quick, Easy, Cheap, Effective, Rugged, and Safe) for extraction of mycotoxins including aflatoxins from food samples in the study developed by Desmarchelier et al. (2010). Both methods showed high extraction efficiency in a broad range of cereal-based products and with a comparable sensitivity. Nevertheless, the easiness-to-handle of these extraction methods was definitely in favor of the QuEChERS-like procedure, avoiding any tedious preparation of extraction cells, requiring less reagents and glassware and involving less intermediate steps. Consequently, a higher sample throughput was possible, with up to 40 individual samples extracted over one working day as compared to the 24 individual samples processed over one and a half working days by the ASE procedure. On a routine basis, the QuEChERS-like method constitutes undeniably the best option. Solid-phase extractions have been used for mycotoxins’ extraction from different kinds of samples. Solid-phase microextraction (SPME) was used by Nonaka et al. (2009). The authors optimized the on-line in-tube SPME-LC-MS and concluded that using this approach it’s possible to continuously extract aflatoxins from samples extracts with no requirement of any other pretreatments, which can then be analyzed by LC–MS. This method is automatic, simple, rapid, selective, and sensitive, and may be easily applied to the analysis of various food samples. Solid-phase extraction (SPE) has also been applied for many years to mycotoxins analysis, once this technique enables the extraction, preconcentration and purification in one step (Alcaide-Molina et al., 2009). 3.2 Clean-up Due to the large number of co-extractives that are present in the sample extracts, most matrices are unsuitable for direct chromatographic analysis, needing a clean-up step. Some studies, according to the detection technique that will be employed only uses the dilution approach to reduce the matrix interferences, as we could observe in the work developed by Acharya & Dhar (2008). The authors describe a simple approach for performing broad-specific noncompetitive immunoassays for the determination of total aflatoxins (AFB1 +AFB2 +AFG1 +AFG2). Twenty grams sample were extracted with 100 mL MeOH:H2O (70:30, v/v) and stirred for 0.5 h. Extracts were filtered through a filter paper.

422

Aflatoxins – Biochemistry and Molecular Biology

The matrix interferences were eliminated by diluting the sample 10-fold with the assay buffer. The most employed clean-up methods in some laboratories are the solid-phase extraction, multifunctional columns or immunoaffinity columns (IACs) (Bacaloni et al., 2008; Huang et al., 2010; Piermarini et al., 2009; Reiter et al., 2010). IACs in combination with HPLC are increasingly used nowadays as reference methods and allow a sufficient elimination of matrix interferences, due to their high selectivity. The immunoaffinity is based on the binding of the immobilized specific antibodies on the surface of a column (Shepard, 2009). Clean-up only with solvents is rarely found nowadays (Sheibani & Ghaziaskar, 2009). The advantages of IACs are the effective and specific extract purification provided, the economic use of organic solvents and the improved chromatographic performance achieved with cleaner samples (Shepard, 2009). The clean-up step has an important role in the quantification techniques, avoiding false positives, allowing better recoveries and helping with the time-life of the equipments. 3.3 Separation and detection Different techniques have been found for the determination of aflatoxins in the last years. Techniques based on ELISA detection (Li et al., 2009), electrochemical sensor (Tan et al., 2009), immunoassays (Saha et al., 2007), Liquid Chromatography tandem Mass Spectrometry (LC-MS) (Kokkonen & Jestoi, 2009; Rubert et al., 2010), Liquid Chromatography with Fluorescence Detection (LC-FLD) (Ibáñez-Vea et al., 2011), Liquid chromatography with ultraviolet detection (Fu et al., 2008) and adsorptive stripping voltammetry (Hajian & Ensafi, 2009) are found in the literature. Aflatoxins separation has been performed for many years by HPLC, using mainly reversedphase columns, with mobile phases composed of water, methanol and acetonitrile mixtures. Chromatographic performance has improved with column technology, particularly with reduced size of the column packing material (Shepard, 2009). Researches employing the Ultra-Performance Liquid Chromatography (UPLC) have brought lower run times and better peak shapes. Huang et al. (2010) employed the UHPLC-MS/MS for the separation and detection of aflatoxins after an extraction with acetonitrile and water and a clean-up with SPE, reaching limits of quantification between 0.012 and 0.073 µg kg-1. The total run time for the separation of AFB1, AFB2, AFG1, AFG2, AFM1 and AFM2 was less than 9 min. The AFs are named due to their properties under UV-irradiation, where AFB1 and AFB2 emit blue fluorescence (350 nm), AFG1 and AFG2 green fluorescence (350 nm). These important features can be used for rapid identification and detection (Reiter et al., 2009). So, although aflatoxins are naturally strongly fluorescent compounds, making them ideal subjects for fluorescence detection, various analogues exhibit solvent-dependent quenching in HPLC solvent systems. In the aqueous mixtures used for reversed-phase chromatography, the fluorescence of AFB1 and AFG1 are significantly quenched (Shepard, 2009). This is generally overcome by some derivatization procedure. In the last years works employing post-column derivatization have been found. Ariño et al. (2009) determined AFB1, AFB2, AFG1 and AFG2 with liquid chromatography using post-column photochemical derivatization for improved sensitivity and selectivity. This technique allowed a fluorescence enhancement about 30 times for aflatoxin B1 and G1. Results showed that post-column photochemical derivatization of aflatoxins increased detectability and selectivity of responses for the LC–FLD system. The average recovery was between 84 and 91%, and LOQ was 0.1 µg kg-1.

Aflatoxins: Contamination, Analysis and Control

423

The coupling of HPLC to mass spectrometry is the more commonly employed detection technique in the last years. The ionization sources employed based on atmospheric pressure ionization techniques such as electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI) has resulted in a range of new methods (Beltrán et al., 2011; Cavaliere et al., 2007; Kokkonen & Jestoi, 2009; Sulyok et al., 2007). The advantages of LC-MS techniques lie in the improved detection limits, the confirmation provided by mass spectral fragmentation and the ability to filter out by mass any impurities that interfere in spectrophotometric detectors. For the determination of 32 mycotoxins, including aflatoxins, in beer, Zachariasova et al. (2010), developed a study with the aim of optimize a simple and high-throughput method. For determination of analytes, ultra-high-performance liquid chromatography hyphenated with high-resolution mass spectrometry utilizing an orbitrap (U-HPLC–orbitrapMS) or time-of-flight (TOFMS) technology was used. Because of significantly better detection capabilities of the orbitrap technology, the U-HPLC– orbitrapMS method was chosen. The U-HPLC–orbitrapMS technology represents a progressive alternative equivalent to MS/MS. The U-HPLC–orbitrapMS system used within this study operates in APCI mode enabled rapid determination of trace levels of multiple mycotoxins potentially occurring in beer samples. Relating to the source of ionization, for aflatoxin determination we have found more studies employing the ESI as source of ionization. Atmospheric pressure photoionization (APPI) is the latest interface introduced in the field of soft ionization techniques, and it was employed in the study developed by Capriotti et al. (2010). Using APPI, detection limits for the investigated compounds were lower than by using ESI, due to a much lower noise and matrix effect. For aflatoxins, LOQs between 0.1 and 0.5 µg kg-1 were reached. The application of aflatoxin-specific antibodies has produced a range of immunoassay analytical methods (Acharya & Dhar, 2008; Li et al., 2009; Saha et al., 2007). A number of commercial enzyme-linked immunosorbent assays (ELISAs) are well established and available. The essential principle of these assays is the immobilization on a suitable surface of antibody or antigen and the establishment of a competitive process involving this resource and components of the analytical solution (Shepard, 2009). Piermarini et al. (2009) developed a method, called ELIME-array (Enzyme-Linked-Immuno-MagneticElectrochemical-array) for the determination of AFB1 in corn samples. In order to determine AFB1 at a level of regulatory relevance, a sample treatment that employs extraction, cleanup and concentration steps, was selected. The recovery of the ELIME-array was calculated by analyzing replicates of four certificate reference materials (CRMs). The method showed recoveries between 95 and 114% with a LOQ of 1.5 ng mL-1. 3.3.1 Matrix effect Another special issue about the determination of contaminants, such as aflatoxins in a variety of samples is the matrix effect. Mainly related to the mass spectrometric techniques, the matrix effect is known as the change of ionization efficiency for the studied analytes in the presence of other compounds (Kruve et al., 2008). Relating to this topic some procedures could be done to guarantee the trueness of the results, avoiding false positives. For aflatoxins’ determination the approaches observed were: dilution, matrix-matched calibration, standard addition and use of internal standard. Some studies employ the AFM1 as I.S, and in others the use of a deuterated one (13C17-AFB1) was observed. The sample clean-up, many times is enough to avoid the matrix effects, but in other cases not.

424

Aflatoxins – Biochemistry and Molecular Biology

3.4 Analytical criteria Some performance criteria are important for obtaining reliable results for aflatoxins’ determination. Table 1 shows a summary of some manuscripts published after 2007, showing which aflatoxins were determined, kind of sample, sample preparation, clean-up, matrix effect, detection, limit of quantification and recoveries. Sample preparation (sample mass, type and volume of extractor solvent)

Clean-up

Matrix Effect Detection

LOQ

R%

Reference

corn

20 g 100 mL MeOH:H2O (70:30, v/v)

-

Dilution 10fold to broad-specific eliminate the noncompetitive immunoassay matrix interferents

5 µg kg-1 (LOD)

86-100

Acharya & Dhar, 2008

Olive leaves and drupes

Automatic SPE 5g 25 mL MeOH:H2O (70:30, v/v)

Automatic SPE

Matrixmatched calibration

LC-ESI-MS/MS

0.03 – 0.11 96-102 µg kg-1

AlcaideMolina et al., 2009

spices

1g 10 mL ACN ultrasonic bath (30 min)

-

Matrixmatched calibration

LC-ESI-MS/MS

1-20 µg kg-1

Amate et al., 2010

pistachios

10 g 1 g NaCl 40 mL MeOH:H2O (8:2, v/v) 20 mL hexane

Immunoafinity column

LC-FLD post-column photochemical derivatization

0.1 µg kg-1 84-91

Ariño et al., 2009

Matrixmatched calibration and internal standard

LC-ESI-MS/MS

0.04 – 0.07 91-102 µg kg-1

Bacaloni et al., 2008

immunoaffinity column

Cleanup eliminated the matrix effect

UHPLC-ESIMS/MS

0.003 0.025 µg kg-1

79 - 112

Beltrán et al., 2011

Immunoaffinity column

-

HPLC-FLD

0.02 - 0.2 µg kg-1

86-96

Brera et al., 2011

LC-APPIMS/MS

0.1 – 0.5 µg kg-1

86-104

Capriotti et al., 2010

LC-ESI-MS/MS

0.04. -0.12 92-107% µg kg-1

Cavaliere et al., 2007

HPLC-FLD

0.03-0.45 µg kg-1

Cho et al., 2008

Aflatoxins Matrix total aflatoxins AFB1, AFB2, AFG1, AFG2 AFB1, AFB2 AFG1, AFG2 AFB1, AFB2 AFG1, FG2 AFB1, AFB2 AFG1, AFG2

AFB1, AFB2 AFG1, hazelnuts AFG2 AFM1 (IS) AFB1, AFB2 Baby food AFG1, and milk AFG2 AFM1

AFB1, AFB2 AFG1, AFG2

AFB1, AFB2 AFG1, AFG2 AFM1 AFB1, AFB2 AFG1, AFG2 AFM1 (I.S)

AFB1, AFB2 AFG1, AFG2

Baby food and paprika

Cereals Wheat and maize samples

1g SPE 20 mL ACN: H2O (80:20, v/v). (Carbograph-4) Ultrasonic bath (10 min) Cereals infant formula - 5 g 20 mL ACN:H2O (80:20, v/v) Liquid samples - 8 g 32 mL ACN Baby food - 50 g 5 g NaCl 250 mL MeOH:H2O (80:20, v/v). Paprika - 25 g 2.5 g NaCl 100 mL MeOH:H2O (80:20, v/v) 1g 6 mL CH3COCH3:H2O:CH3COOH (80:19:1, v/v/v) ultrasonic bath (20 min)

-

Olive oil

MSPD (C18) 0.32 g 6 mL MeOH:H2O (80:20, v/v)

-

Curry Red pepper paste Ginger product Red pepper flour Black pepper Cinnamon powder

25 g 100 mL MeOH:H2O (70:30, v/v) 1% NaCl

Immunoafinity column

Matrixmatched calibration

100-139

68.1-103.9

425

Aflatoxins: Contamination, Analysis and Control

Aflatoxins Matrix

AFB1, AFB2 AFG1, AFG2 AFB1, AFB2 AFG1, AFG2 AFM1 AFB1, AFB2 AFG1, AFG2 AFB1, AFB2 AFG1, AFG2 AFB1, AFB2 AFG1, AFG2 AFB1, AFB2 AFG1, AFG2 AFB1, AFB2 AFB1, AFB2 AFG1, AFG2 AFM1, AFM2 AFB1, AFB2 AFG1, AFG2 AFM, AFM2 AFB1, AFB2 AFG1, AFG2 AFB1, AFB2 AFG1, AFG2 AFB1, AFB2 AFG1, AFG2 AFB1 AFB1, AFB2 AFG1, AFG2 AFB1, AFB2 AFG1, AFG2

maize, wheat, rye, rice, oat, barley, soya, and infant cereals Maize Walnuts Biscuits Breakfasts cereals Corn peanuts

Sample preparation (sample mass, type and volume of extractor solvent)

Clean-up

Matrix Effect Detection

LOQ

R%

QuEChERS - 5 g 10 mL ACN 0.5% CH3COOH ASE - 5 g ACN:H2O:CH3COOH (80:19:0.5, v/v/v)

defatting step with n-hexane

Standard addition

LC-ESI-MS/MS

1.0 – 2.0 µg kg-1

QuEChERS 89-116 Desmarchelier ASE et al., 2010 67-107

5g 10 mL ACN:H2O (80:20, v/v) biscuit samples - 20 mL ACN:H2O (80:20, v/v) -

-

UHPLCMS/MS

Matrix-marched 0.03-3.5 µg 71.3-104-7 calibration kg-1

25 g 80 mL ACN:H2O (84:16, v/v)

Immunoafinnity column

UPLC-UV

0.63-1.07 µg kg-1

83.4-94.7

Fu et al., 2008

immunoaffinity column

-

HPLC-FLD

0.08-0.16 µg kg-1

68.3-87.7

Ghali et al., 2009

immunoaffinity column

-

HPLC-FLD Post columns PHRED

5.0 µg kg-1

52-89

Gnonlonfin et al., 2010

immunoaffinity columns

-

HPLC-UV-FLD

0.1-3.5 ng mL-1

65-90

Gonçalez et al., 2008

-

Diluted 10Adsorptive fold to avoid stripping interferences voltametry

0.1-0.115 ng mL-1 (LOD)

-

Hajian & Ensafi, 2009

10 g 40 mL MeOH: H2O (80:20, v/v) 1 g NaCl 20 mL n-hexane 10 g 1 g NaCl Cassava flour 25 mL MeOH:H20 (80:20, v/v) 25 g 5 g NaCl Peanuts 125 mL MeOH:H20 (7:3 v/v) 5g 10 mL MeOH:H20 Groundnut (80:20, v/v) 5 mL hexane Sorghum pistachios

Reference

Frenich et al., 2009

Traditional Chinese medicines

2g 10 mL ACN:H2O (84:16, v/v)

SPE

Internal standard [13C17]-AFB1

UHPLC-ESIMS/MS

0.1-0.39 µg 85.6-117.6 kg-1

Han et al., 2010

Peanuts and their derivative products

2.5 g 10 mL ACN:H2O (84:16, v/v)

SPE

Matrixmatched calibration

UHPLC-ESIMS/MS

0.0120.273 µg kg-1

Huang et al., 2010

barley

10 g 50 mL ACN:H2O (60:40, v/v)

immunoaffinity column

Automatic ASE 10 g sample Extraction with acetonitrile

-

Cereals Wheat Barley Oats

0.5 g nuts, cereals, 1 mL MeOH:H20 dried fruits, (80:20, v/v) and spices SPME 25 g Corn 100 mL ACN:H2O (84:16, v/v) 2g 10 mL of Cereal flours MeOH:PB1 (80:20, v/v) Ultrasonic bath (20 min) SPME rice

50 g 100 mL MeOH:H20 (80:20, v/v)

Mycosep columns

UHPLC-FLD

0.038 0.15 µg kg- 71.7-99.6 1

Ibáñez-Vea et al., 2011

Matrixmatched calibration

LC-ESI-MS/MS

20 – 65 µg kg-1

61-94

Kokkonen & Jestoi, 2009

AFM1 (I.S.)

LC-ESI-MS

2.1 - 2.8 pg mL-1 (LOD)

80.8-109.1

Nonaka et al., 2009

Matrixmatched calibration

ELIME-array

1.5 ng mL-1

95-114%

Piermarini et al., 2009

0.1-0.63 µg kg-1

49-59

Quinto et al., 2009

0.44-0.6 µg kg-1

83-102

Reiter et al., 2010

Immunoafinity column

immunoaffinity columns

74.7-86.8

HPLC-FLD

426

Aflatoxins – Biochemistry and Molecular Biology

Aflatoxins Matrix

Sample preparation (sample mass, type and volume of extractor solvent)

AFB1, AFB2 AFG1, AFG2

cereals

MSPD (1 g C18) 1g 10 mL ACN

AFB1

Chili

2g 5 mL MeOH:H20 (80:20, v/v)

AFB1, AFB2 AFG1, AFG2

Tigernuts and Their Beverages

MSPD (2 g C18) 1 g or 1 mL 10 mL hexane 10 mL ACN

AFB1, AFB2

pistachio

PFE 7g 5 mL n-hexane MeOH:H20 (80:20, v/v)

purified with chloroform

AFB1, AFB2, AFG1, AFG2

paprika

25 g 100 mL of MeOH:H20 (60:40, v/v)

immunoaffinity column

-

HPLC-FLD

-

Matrix matched calibration

HPLC/ ESI-MS/MS

AFB1, AFB2 AFG1, AFG2 AFM1 AFB1 AFB1, AFB2 AFG1, AFG2 AFB1, AFB2 AFG1, AFG2

bread, fruits, vegetables, 0.5 g jam, 2 mL ACN:H2O:CH3COOH cheese, (79:20:1, v/v/v) chestnuts red wine 1g Rice 5 mL MeOH:H20 (80:20, v/v) beer

Clean-up

Matrixmatched calibration

4 mL beer 16 mL ACN

wheat flour, 50 g corn flour, 250 mL MeOH:H20 poultry feeds (80:20, v/v)

Matrix Effect Detection

immunoaffinity column

LOQ

R%

Reference

LC-ESI-MS/MS 1 µg kg-1

64-91

Rubert et al., 2010

Membranebased immunoassay

2 µg kg-1

88-101

Saha et al., 2007

LC-FLD

0.21-1.49 µg kg-1 tigernuts 0.13-0.57 µg L-1 beverages

72.3-82.1 (tigernuts) Sebastià et al., 74.0-86.3 2010 (beverages)

100

Sheibani & Ghasiaskar, 2009

0.23-0.45 µg kg-1

75.6-108

Shundo et al., 2009

0.7-1.5 µg kg-1 (LOD)

97-100

Sulyok et al., 2007

Electrochemical 0.1 µg L-1 (LOD) sensor

88.5-112

Tan et al., 2009

Matrix matched calibration

U-HPLC– orbitrapMS

0.5 – 3.0 µg L-1

90-117

Zachariasova et al., 2010

-

LC- FLD

0.01 – 0.01 >65% µg kg-1

HPLC-FLD

Zinedine et al., 2007

Table 1. Main parameters about extraction and determination of aflatoxins from 2007 to the present. 3.5 Conclusions and analysis tools of tomorrow Determination of aflatoxins has been carried out using TLC, HPLC, LC–MS, LC–MS–MS, and immunological methods. Each one of the techniques has advantages and disadvantages. TLC provides an economical screening method. HPLC methods coupled with fluorescence detection are sensitive and the most widely used methods, but most require a derivatization step. Immunoassays provide rapid screening for total aflatoxin, but they may not be sufficiently reliable as quantitative methods for individual aflatoxins. LC–MS methods are specific and sensitive, and their use is becoming increasingly widespread. However, due to the low levels and the number of interferences from the matrices, usually, a sample preparation step is required to allow the extraction, preconcentration, and clean-up, enhancing the sensitivity and selectivity. The advance in the extraction and determination of aflatoxins will continue increasing together with the improvement of analytical science. The search for sample preparation methods that allow fast extraction, good accuracy and precision, low extraction of interferences, low consumption of solvents will continue together with the increase in

Aflatoxins: Contamination, Analysis and Control

427

detection techniques with higher accuracy and sensibility. So, the determination of aflatoxins in foods will continue to be developed and improved.

4. Legislation, desintoxication and control Concern about the potential hazards posed by dietary aflatoxins started in the 1960s after some 100000 turkey poults in Great Britain died as a result of aflatoxin exposure from their feed. When it became evident that aflatoxin exposure caused cancer in many species, most countries, established various regulations for aflatoxin levels (either total aflatoxins or for AFB1) in food and/or feed in order to limit exposure to this group of mycotoxins (Van-Egmond et al., 2007). These initial regulations on aflatoxins were not based on the derivation of a TDI (estimated tolerable daily intake), but rather on a desire to keep levels as low as technologically feasible (basis for regulations in some countries), or ‘free’ of aflatoxins by not allowing residues above the analytical detection limit (basis for regulations in some other countries). The early prudent actions regarding aflatoxins by governments have been justified, since AFB1 has been found to be a potent genotoxic agent and carcinogen in many test systems and animal species (Kuiper-Goodman, 1995; Wogan, 1974). Worldwide, aflatoxins because of their prevalence and toxicity are important in public health. Public health concerns center on both primary poisoning from aflatoxins in commodities, food and feed stuffs, and relay poisoning from aflatoxins in milk. The allowable levels of aflatoxins in animal feedstuff and human foods vary with governmental jurisdictions (Coppock & Christian, 2007). Aflatoxins are of great concern because of their detrimental effects on the health of humans and animals, including carcinogenic, mutagenic, teratogenic and immunosuppressive effects. AFB1 is the most potent hepatocarcinogen known in mammals and is classified by the International Agency of Research on Cancer as Group 1 carcinogen (Eaton & Gallagher, 1994 as cited in Zinedine, 2009). The hazardous nature of aflatoxin to humans and animals has necessitated the need for establishment of control measures and tolerance levels by national and international authorities. Different countries have different regulations for aflatoxin. The general trend is that industrialized countries usually set lower tolerance levels than the developing countries, where most of the susceptible commodities are produced. However, such lack of harmony may give rise to difficulties in the trade of some commodities (Aibara & Maeda, 1989; Ismail, 1997). The first legislative act was undertaken in 1965 by the Food and Drug Administration (FDA) of the USA, which proposed a tolerance level of 30 pg kg-1 of total aflatoxins (Bl + Gl + B2 + G2). With increasing awareness of aflatoxins as potent toxic substances, the proposed level was lowered to 20 pg kg-1 in 1969. The FDA has action levels for aflatoxins regulating the levels and species to which contaminated feeds may be fed (Table 2). In 1973, the European Economic Community (EEC) established legislation on maximum permitted levels of AFBl in different types of feedstuffs. The legislation has been frequently amended since then (EEC, 1974; FDA, 1977; Ismail 1997). The European Community levels are more restrictive (Tables 3 and 4), 4 µg kg-1 total aflatoxin in food for human consumption are the maximum acceptable limits in the EU, the strictest in standard worldwide. Human foods are allowed 4–30 ppb aflatoxin, depending on the country involved (John, 2007).

428

Aflatoxins – Biochemistry and Molecular Biology

Concentration (µg kg-1) 300 300 200

Commodity Cottonseed meal as a feed ingredient Corn and peanut products for finishing beef cattle Corn and peanut products for finishing swine Corn and peanut products for breeding beef cattle, swine and mature poultry Corn for immature animals and dairy cattle All products, except milk, designated for humans All other feedstuffs Milk

100 20 20 20 0.5

Table 2. U.S. Food and Drug Administration action levels for total aflatoxins in food and feed (µg kg-1). Human food

AFB1 (µg kg-1)

AFB1, B2, G1, G2 (µg kg-1)

M1 (µg kg-1)

Groundnuts, dried fruit and processed products thereof

2

4

-

8

15

-

5

10

-

2

4

-

-

0,05

Groundnuts subjected to sorting or physic treating As above but for nuts and dried fruits Cereals (including maize) and processed products thereof Milk

-

Table 3. European Union for aflatoxins in human food (µg

kg-1).

The Brazilian National Agency for Sanitary Vigilance established the Resolution (RDC) nº 7 of February 2011 which provides for the maximum permissible (LMT) for aflatoxins (Table 5) and other mycotoxins in food. Feed

AFB1 (µg kg-1)

Feed

AFB1 (µg kg-1)

Feed (exceptions below)

50

Complete feedstuff for pigs and poultry

20

Groundnuts, copra, palm kernel, cottonseed, babasu, maize and products derived from processing thereof

20

Other complete feedstuffs

10

Complete dairy feed

5

Complete feed for lambs and calves

10

Complementary feedstuffs for cattle, sheep, goats (except dairy, calves and lambs) Complementary feedstuffs for pigs and poultry (except for young animals)

Table 4. European Union regulations for aflatoxins in feeds (µg kg-1).

50

30

429

Aflatoxins: Contamination, Analysis and Control

It is estimated that about 35% of human cancers are directly related to diet, and the presence of aflatoxins in foods is considered an important factor in the formation of liver cancer, mainly in tropical countries. The reduction of population exposure to aflatoxin, and the consequent reduction of health risks will only be possible with a job with the food producers and efficient actions of sanitary vigilance (Doll & Peto, 1981).

Mycotoxin

AFB1, B2, G1, G2

Commodity Cereals and cereal products, except corn and derivatives, including malted barley Beans Chestnuts except Brazil-nut, including walnuts, pistachios, hazelnuts and almonds Dried and dehydrated fruits Brazil-nut shell for direct consumption Brazil-nut shelled for direct consumption Brazil-nut shelled for further processing Cereal-based foods for infant feeding (infants and toddlers) Infant formulas and follow-up formula for infants and toddlers Cocoa beans

10 20 10 15

Cocoa and chocolate

5

Spices: Capsicum spp. (dried fruits, whole or ground, including peppers, chili powder, cayenne and paprika), Piper spp. (the fruit, including white pepper and black pepper) Myristica fragrans (nutmeg) Zingiber officinale (ginger) Curcuma longa (turmeric). Spice mixtures that containing one or more of the spices listed above. Groundnut (in shell), (peeled, raw or roasted), peanut butter or peanut butter. Corn, grain (whole, broken, crushed, ground), flour or corn meal Aflatoxin M1

Maximum limit tolerated (µg kg-1) 5 5 10

1 1 10

20

20 20

Fluid milk

0,5

Milk powder

5

Cheese

2,5

Table 5. Maximum permitted (LMT) for aflatoxin in Brazil. Aflatoxins can be detoxified or removed from contaminated food and nutrients by physical, chemical or biological methods. The inactivation of these compounds by physical and chemical methods have not proved to be effective and economically viable (Mishra & Das, 2003). However, biological degradation offers an attractive alternative to eliminate these

430

Aflatoxins – Biochemistry and Molecular Biology

toxins retaining food nutritional value. In the last decade it became clear that fungi are among the microorganisms that play a major role in mycotoxin degradation in particular AFB1 (Zucchi et. al., 2008). Aflatoxins are thermostable, so the physical treatment by heat results in only small changes in their levels (Tripathi & Mishra, 2010). Chemical treatments using solvents are able to extract these compounds causing minimal effect on nutritional quality, however, this technology is still impractical and expensive, besides inducing odors and flavors. Ammoniation is also used as an effective and practical application for decontamination of agricultural products containing aflatoxins (Allameh et al., 2005). Ozonation is the chemical method that has been most studied for the decontamination of aflatoxins in foods, once ozone has been recognized as safe by the Food and Drug Administration in 2001 (Zorlugenç et al., 2008). Currently, several studies have shown that aflatoxins are susceptible to some microorganisms such as fungi, bacteria and yeasts, being for this reason studied as a form of biological degradation. Taylor et al. (2010) studied some enzymes belonging to the group of actinomicetales specifically Mycobacterium smegmatis which is capable of catalyzing the ester group of aflatoxins by activating the molecules for the spontaneous hydrolysis and subsequent decontamination. Niu et al. (2008) studied several microorganisms from microbial sources that have coumarin as a carbon source. The results indicated that degradation was performed enzymatically by protease. Cacciamani et al. (2007) evaluated AFB1 and ochratoxin A degradation by solid fermentation using A. oryzae and Rhizopus sp. The first showed higher AFB1 decontamination (80%). There are several alternatives for detoxification of aflatoxins in foods, such as the use of acids and bases in the industry, being replaced by processes that involve components such as ozone GRAS and the use of fungi, bacteria or yeasts.

5. References Acharya, D. & Dhar, T. (2008). A novel broad-specific noncompetitive immunoassay and its application in the determination of total aflatoxins. Analytica Chimica Acta, Vol.630, No.1, pp. 82–90, ISSN 0003-2670 Aibara, K.; Maeda, K. (1989). The regulation for aflatoxin in Japan and the current situation of aflatoxin contamination of food imported from throughout the world. In: Mycotoxin and Phycotoxins, S. Natori, K. Hashimoto; Y. Ueno, Elsevier, 978-90-8686007-4, Amsterdam Alcaide-Molina, M.; Ruiz-Jiménez, J.; Mata-Granados, J. & Luque de Castro, M. (2009). High through-put aflatoxin determination in plant material by automated solid-phase extraction on-line coupled to laser-induced fluorescence screening and determination by liquid chromatography–triple quadrupole mass spectrometry. Journal of Chromatography A, Vol.1216, No.7, pp. 1115–1125, ISSN 0021-9673 Allameh, A.; Safamehr, A.; Mirhadi, S.A.M.; Shivazad, M.; Razzaghi-Abyaneh, M. & AfsharNaderi, A. (2005). Evaluation of biochemical and production parameters of broiler chicks fed ammonia treated aflatoxin contaminated maize grains. Animal Feed Science Technology Vol.122, No.3, pp 289-301, INSS 0377-8401 Almeida, A. V. A. F.; Botura, M. B.; Abreu, R. D.; Bittencourt, T. C. C. & Batatinha, M. J. M. (2009). Ocorrência de aflatoxinas em milho destinado à alimentação de aves no

Aflatoxins: Contamination, Analysis and Control

431

estado da Bahia. Arquivos Instituto Biológico, São Paulo, Vol.76, No.3, pp. 353-358, ISSN 0020-3653 Amaral, K. A. S.; Nascimento, G. B.; Sekiyama, B. L.; Janeiro, V.; Machinski Jr., M. (2006). Aflatoxinas em produtos à base de milho comercializados no Brasil e riscos para a saúde humana. Ciência e Tecnologia de Alimentos. Vol.26, No.2, pp. 336-342, ISSN 0101-2061 Amate, C.; Unterluggauer, H.; Fischer, R.; Fernández-Alba, A. & Masselter, S. (2010). Development and validation of a LC–MS/MS method for the simultaneous determination of aflatoxins, dyes and pesticides in spices. Analytical and Bioanalytical Chemistry, Vol.397, No.1, pp. 93–107, ISSN 1618-2650 Ariño, A.; Herrera, M.; Estopañan, G.; Rota, M.; Carramiñana, J.; Juan, T. & Herrera, A. (2009). Aflatoxins in bulk and pre-packed pistachios sold in Spain and effect of roasting. Food Control, Vol.20, No.9, pp. 811–814, ISSN 0956-7135 Bacaloni, A.; Cavaliere, C.; Cucci, F.; Foglia, P.; Samperi, R. & Laganà, A. (2008) Determination of aflatoxins in hazelnuts by various sample preparation methods and liquid chromatography–tandem mass spectrometry. Journal of Chromatography A, Vol.1179, No.2, pp. 182–189, ISSN 0021-9673 Beltrán, E.; Ibáñez, M.; Sancho, J.; Cortés, M.; Yusà, V. & Hernández, F. (2011). UHPLC– MS/MS highly sensitive determination of aflatoxins, the aflatoxin metabolite M1 and ochratoxin A in baby food and milk. Food Chemistry, Vol.126, No.2, pp. 737–744. ISSN 0308-8146 Bircan, C. (2009). Incidence of ochratoxin A in dried fruits and co-occurrence with aflatoxins in dried figs. Food and Chemical Toxicology, Vol.47, No.8, pp.1996–2001, ISSN 0286915 Boutrif, E. (1998). Prevention of Aflatoxin in Pistachios, 17.01.2011, Available from

Brera, C.; Debegnach, F.; Santis, B.; Pannunzi, E.; Berdini, C.; Prantera, E.; Gregori, E. & Miraglia, M. (2011). Simultaneous determination of aflatoxins and ochratoxin A in baby foods and paprika by HPLC with fluorescence detection: A single-laboratory validation study. Talanta, Vol.83, No.5, pp. 1442–1446, ISSN 0039-9140 Cacciamani J. M.; Peres G. L.; Garda-Buffon J. & Badiale-Furlong E. (2007). Efeito dos tratamentos térmicos seco e úmido nos níveis de aflatoxina B1 e ocratoxina A presentes em farelo e farinhas cereais. Boletim do Centro de Pesquisa de Processamento de Alimentos, Vol.25, No.1, pp. 157-164, ISSN 19839774 Cano-Sancho, G.; Marin, S.; Ramos, A. J.; Peris-Vicente, J. & Sanchis, V. (2010). Occurrence of aflatoxin M1 and exposure assessment in Catalonia (Spain). Revista Iberoamericana de Micología, Vol.27, No.3, pp. 130-135, ISSN 1130-1406 Capriotti, A.; Foglia, P.; Gubbiotti, R.; Roccia, C.; Samperi, R.; & Laganà, A. (2010) Development and validation of a liquid chromatography/atmospheric pressure photoionization-tandem mass spectrometric method for the analysis of mycotoxins subjected to commission regulation (EC) No. 1881/2006 In cereals. Journal of Chromatography A, Vol.1217, No.39, pp. 6044–6051, ISSN 0021-9673 Carvajal, M.; Bolonos, A.; Rojo, F. & Mendez, I. (2003). Aflatoxin in pasteurized and ultrapasteurized milk with different fat content in Mexico. Journal of Food Protection. Ames, Vol.66, No.10, pp. 1885-1892, ISSN 0362-028X

432

Aflatoxins – Biochemistry and Molecular Biology

Cavaliere, C.; Foglia, P.; Guarino, C.; Nazzari, M.; Samperi, R. & Laganà, A. (2007). Determination of aflatoxins in olive oil by liquid chromatography–tandem mass spectrometry, Analytica Chimica Acta, Vol.596, No.1, pp. 141–148, ISSN 0003-2670 Cho, S.; Lee, C.; Jang, M.; Son, Y.; Lee, S.; Choi, I.; Kim, S. & Kim, D. (2008). Aflatoxins contamination in spices and processed spice products commercialized in Korea. Food Chemistry, Vol.107, No.7, pp. 1283-1288, ISSN 0308-8146 Coelho, C.S.; Almeida, T.L. & Badiale-Furlong, E. (1999). Migração de micotoxinas durante o processo de parboilização do arroz. Brazilian Journal Food Technology, Vol.2, No.2, pp. 43-50, ISSN 1516-7275 Coppock, W.R. & Christian, R.G. (2007). Aflatoxins, In: Veterinary Toxicology – Basic and Clinical Principles, R. C. Gupta; pp. 939-950, Academic Press, ISBN 0123704677, San Diego. D’Angelo, A.; Bellino, C.; Alborali, G. L.; Biancardi, A.; Borelli, A.; Capucchio, M. T.; Maurella, C. & Cagnasso, A. (2007). Neurological signs associated with aflatoxicosis in Piedmontese calves. Veterinary Record, Vol.160, No.20, pp. 698-700, ISSN 00424900 Dashti, B.; Al-Hamli, S.; Alomirah, H.; Al-Zenki, S.; Abbas, A.B. & Sawaya, W. (2009). Levels of aflatoxin M1 in milk, cheese consumed in Kuwait and occurrence of total aflatoxin in local and imported animal feed. Food Control, Vol.20, No.7, pp. 686-690, ISSN 0956-7135 Dekkers, S.; Van Der Fels-Klerx, H.J.; Jeurissen, S.M.F.; Kandhai, M. C.; Booij, C.J.H. & Bos, P.M.J. (2008). Development of a model to assess the occurrence of mycotoxins in wheat, maize and nuts, A holistic approach, Report 320111002, National Institute for Public Health and the Environment, Bilthoven, The Netherlands, 2008, 87 pp., Available from http://www.rivm.nl/bibliotheek/rapporten/320111002.pdf Desmarchelier, A.; Oberson, J.; Tella, P.; Gremaud, E.; Seefelder, W. & Mottier, P. (2010). Development and comparison of two multiresidue methods for the analysis of 17 mycotoxins in cereals by liquid chromatography electrospray ionization tandem mass spectrometry. Journal of Agricultural and Food Chemistry, Vol.58, No.13, pp. 7510-7519, ISSN 0021-8561 Dilkin, P. (2002). Micotoxicose suína: aspectos preventivos, clínicos e patológicos. Biológico. Vol. 64 , No. 2, pp. 187-191, ISSN 1980 - 6221 Doll, R. & Peto, R. (1981). The causes of câncer: quantitative estimates of avoidable risks of câncer in the United States today. Journal of the National Cancer Institute. Vol.66, No.6, pp. 291-308, ISSN 0027-8874 Dombrink-Kurtzman, M.A. (2008). Economic Aspects of Mycotoxins in Fruits and Vegetables, In: Mycotoxins in Fruits and Vegetables, Barkai-Golan, R. & Nachman, P., pp. 27-44, Academic Press, ISBN 978-0-12-374126-4, New York Dors, G.C.; Pinto, L.A.A. & Badiale-Furlong, E. (2009). Migration of mycotoxins into rice starchy endosperm during the parboiling process. LWT - Food Science and Technology, Vol.42, No.1, pp. 433-437, ISSN 0023-6438 Dors, G.C.; Bierhals, V.S. & Badiale-Furlong, E. (2011). Parboiled rice: chemical composition and the occurrence of mycotoxins. Ciência e Tecnologia de Alimentos, Vol.31, No.1, pp. 172-177, ISSN 0101-2061 Drusch, S. & Ragab, W. (2003) Mycotoxins in fruits, fruit juices, and dried fruit. Journal of Food Protection, Vol.66, No.8, pp. 1514–1527, ISSN 0362-028X

Aflatoxins: Contamination, Analysis and Control

433

EEC (1974). Council Directive 74/63/EEC on the fixing of maximum permitted levels for undesirable substances and products in feedingstuffs. Official Journal of the European Community, No. L 38 Ellis, W.O.; Smith, P.J.; Simpson, B.K.; Khanizadeh, S. & Oldham, J. H. (1993). Control of Growth and Aflatoxin Production of Aspergillus flavus under Modified Atmosphere Packaging Conditions. Food Microbiology, Vol.10, No.1, pp. 9-21, ISSN 0740-0020 Fallah, A.A.; , Jafarib, T., Fallahc A. & Rahnamad M. (2009). Determination of aflatoxin M1 levels in Iranian white and cream cheese. Food and Chemical Toxicology. Vol.47, No.8, pp. 1872-1875, ISSN 0278-6915 Fallah, A.A. (2010). Levels of aflatoxin M1 in milk, cheese consumed in Kuwait and occurrence of total aflatoxin in local and imported animal feed. Food and Chemical Toxicology. Vol.48, No.3, pp. 988-991, ISSN 0278-6915 FDA (1977). FDA Proposed Guidelines for Aflatoxin. Cereal Foods World, 22, 532-533, 541. Frenich, A.; Vidal, J.; Romero-González, R. & Aguilera-Luiz, M. (2009). Simple and highthroughput method for the multimycotoxin analysis in cereals and related foods by ultra-high performance liquid chromatography/tandem mass spectrometry. Food Chemistry, Vol.117, No.4, pp. 705–712, ISSN 0308-8146 Fu, Z.; Huang, X. & Min, S. (2008). Rapid determination of aflatoxins in corn and peanuts, Journal of Chromatography A, Vol.1209, No.1, pp. 271–274, ISSN 0021-9673 Ghali, R.; Belouaer, I.; Hdiri, S.; Ghorbel, H.; Maaroufi, K. & Hedilli, A. (2009). Simultaneous HPLC determination of aflatoxins B1, B2, G1 and G2 in Tunisian sorghum and pistachios. Journal of Food Composition and Analysis, Vol.22, No.7/8, pp. 751–755. ISSN 0889-1575 Giorni, P.; Battilani, P.; Pietri, A. & Magan, N. (2008). Effect of Aw and CO2 level of Aspergillus flavus growth and aflatoxin production in high moisture maize postharvest. International Journal of Food Microbiology, Vol.122, No.1-2, pp. 109-113, ISSN 0168-1605 Gnonlonfin, G.; Katerere, D.; Adjovi, Y.; Brimer L.; Shephard, G. & Sanni, A. (2010). Determination of aflatoxin in processed dried cassava root: validation of a new analytical method for cassava flour. Journal of AOAC International, Vol.93, No.6, pp. 1882-1887, ISSN 1944-7922 Gonçalez, E.; Nogueira, J.; Fonseca, H.; Felicio, J.; Pino, F. & Corrêa, B. (2008). Mycobiota and mycotoxins in Brazilian peanut kernels from sowing to harvest. International Journal of Food Microbiology, Vol.123, No.3, pp. 184–190, ISSN 0168-1605 Hajian, R. & Ensafi, A. (2009). Determination of aflatoxins B1 and B2 by adsorptive cathodic stripping voltammetry in groundnut. Food Chemistry, Vol.115, No.3, pp. 1034–1037. ISSN 0308-8146 Han, Z.; Zheng, Y.; Luan, L. Cai, Z.; Ren, Y. & Wu, Y. (2010). An ultra-high-performance liquid chromatography-tandem mass spectrometry method for simultaneous determination of aflatoxins B1, B2, G1, G2, M1 and M2 in traditional Chinese medicines. Analytica Chimica Acta, Vol.664, No.2, pp. 165–171. ISSN 0003-2670 Herzallah, S.M. (2009). Determination of aflatoxins in eggs, milk, meat and meat products using HPLC fluorescent and UV detectors. Food Chemistry, Vol.114, No.3, pp. 1141– 1146, ISSN 0308-8146

434

Aflatoxins – Biochemistry and Molecular Biology

Heshmati, A. & Milani, J. M. (2010). Contamination of UHT milk by aflatoxin M1 in Iran. Food Control, Vol.21, No.1, pp. 19-22, ISSN 0956-7135 Huang, B.; Han, Z.; Cai, Z.; Wub, Y. & Ren, Y. (2010). Simultaneous determination of aflatoxins B1, B2, G1, G2, M1 and M2 in peanuts and their derivative products by ultra-high-performance liquid chromatography–tandem mass spectrometry. Analytica Chimica Acta, Vol.662, No.1, pp. 62–68, ISSN 0003-2670 Ibáñez-Vea, M.; Corcuera, L.; Remiro, R.; Murillo-Arbizu, M.; González-Peñas, E. & Lizarraga, E. (2011). Validation of a UHPLC-FLD method for the simultaneous quantification of aflatoxins, ochratoxin A and zearalenone in barley. Food Chemistry, Vol.127, No.1, pp. 351–358, ISSN 0308-8146 Institute of Food Science and Technology Trust Fund. (2006). Mycotoxins, Information Statement, 02.03.2011, Available from

Ismail Y.S.R. (1997). Aflatoxin in food and feed: occurrence, legislation and inactivation by physical methods. Food Chemistry, Vol.59, No.1, pp. 57-67, ISSN 0308-8146 John, L.R. (2007). Some major mycotoxins and their mycotoxicoses – an overview. International Journal of Food Microbiology, Vol.119, No.1-2, pp. 3-10, ISSN 0168-1605 Khayoon, W.S.; Saad B.;, Yan C. B.; Hashim N. H.; Ali A. S. M., Salleh M. I. & Salleh B. (2010). Determination of aflatoxins in animal feeds by HPLC with multifunctional column clean-up . Food Chemistry, Vol.118, No.3, pp. 882-886, ISSN 0308-8146 Klich, M. Mendoza, C. Mullaney, E. Keller, N. & Bennett, J.M. (2001). A new sterigmatocystin-producing Emericella Variant from agricultural desert soils. Systematic and Applied Microbiology, Vol.24, No.1, pp. 131-138, ISSN 0723-2020 Kokkonen, M. & Jestoi, M. (2009). A Multi-compound LC-MS/MS Method for the Screening of Mycotoxins in Grains. Food Analytical Methods, Vol.2, No.2, pp. 128–140, ISSN 1936- 9751 Kruve, A.; Künnapas, A.; Herodes, K. & Leito, I. (2008) Matrix effects in pesticide multiresidue analysis by liquid chromatography–mass spectrometry. Journal of Chromatography A, Vol.1187, No.1/2, pp. 58–66, ISSN 0021-9673 Kuiper-Goodman, T. Mycotoxins: risk assessment and legislation. (1995) Toxicology Letters, Vol.82-83, (Dec, 1995), pp. 853-859, ISSN 0378-4274 KWIATKOWSKI, A. & ALVES, A.P.F. (2007). Importância da detecção e do controle de aflatoxinas em alimentos. SaBios- Revista Saúde e Biologia, Vol.2, No.2 pp. 45-54, ISSN 1980-0002 Li, P.; Zhang, Q.; Zhang, W.; Zhang, J.; Chen, X.; Jiang, J.; Xie, L. & Zhang, D. (2009). Development of a class-specific monoclonal antibody-based ELISA for aflatoxins in peanut. Food Chemistry, Vol.115, No.1, pp. 313–317, ISSN 0308-8146 Luchese, R.H. & Harrigan, W.F. (1993). Biosynthesis of Aflatoxin – the Role of Nutritional Factors. Journal of Applied Bacteriology, Vol.74, No.1, pp. 5–14, ISSN 0021-8847 Manetta, A.C.; Giammarco, M.; Di Giuseppe, L.; Fusaro, I.; Gramenzi, A.; Formigoni, A.; Vignola, G.; Lambertini, L. (2009) Distribution of aflatoxin M1 during Grana Padano cheese production from naturally contaminated milk. Food Chemistry, Vol.113, No.2, pp. 595-599, ISSN 0308-8146 Martins, M.L. & Martins, H.M. (2004). Aflatoxin M1 in yoghurts in Portugal. International Journal of Food Microbiology, Vol. 91, No.3, pp. 315-317, ISSN 0168-1605

Aflatoxins: Contamination, Analysis and Control

435

Marques, P.J.L. (2007). Avaliação de aflatoxina e zearalenona em quirera (canjiquinha de milho) na região dos Campos Gerais. Dissertação apresentada para obtenção do título de mestre em Ciência e Tecnologia de Alimentos, Universidade Estadual de Ponta Grossa – PR Marvin, H.J.P.; Kleter, G.A.; Prandini, A.; Dekkers, S. & Bolton, D.J. (2009a). Early identification systems for emerging foodborne hazards, Food and Chemical Toxicology, Vol.47, No.5, pp. 915–926, ISSN 0278-6915 Marvin, H.J.P.; Kleter, G.A.; Frewer, L.J. S.; Cope, M.T.A. & Wentholt, G. (2009b). Rowe, Aworking procedure for identifying emerging food safety issues at an early stage: implications for European and international risk management practices, Food Control, Vol. 20, No. 2, pp. 345–356, ISSN 0956-7135 Matumba, L.; Monjerezi, M.; Khonga, E.B. & Lakudzala D.D. (2010). Aflatoxins in sorghum, sorghum malt and traditional opaque beer in southern Malawi. Food Control, Vol.22, No.2, pp. 266-268, ISSN 0956-7135 Mclean, M. & Dutton, M.F. (1995). Cellular Interations and Metabolism of Aflatoxin: an Update. Farmacology Therapeutics, Vol.65, No.2, pp. 163-192, ISSN 0163-7258 D’Mello, J.P.F. (2003). Mycotoxins in Cereal Grains, Nuts and Other Plant Products. International. Food Safety: Contaminants and Toxins, pp. 65-90, CABI, ISBN 0-85199607-8, Wallingford Mishra, H.N. & Das, C. A review on biological control and metabolism of aflatoxin. (2003) Critical Reviews in Food Science and Nutrition, Vol.43, No.3, pp. 245–264, INSS 10408398 Molina, M. & Giannuzzi, L. (2002). Modelling of Aflatoxin Production by Aspergillus parasiticus in a Solid Medium at Different Temperatures, pH and Propionic Acid Concentrations. Food Research International, Vol.35, No.6, pp. 585-594, ISSN 09639969 Montagna, M.T; Napoli, C.; De Giglio, O.; Latta, R. & Barbutti, G. (2008). Occurrence of aflatoxin M1 in Dairy products in Southem Italy. International Journal Molecular Sciences. Basel, Vol.9, No.12, pp. 2614-2621, ISSN 1422-0067 Nakai, V.K.; Rocha, L.O.; Gonçalez, E.; Fonseca, H.; Ortega, E.M.M. & Corrêa, B. (2008) Distribution of fungi and aflatoxins in a stored peanut variety. Food Chemistry Vol.106, No.1, pp. 285–290, ISSN 0308-8146 Niu, T.; Guan, S.; Ji, C.; Zhou, T.; Li, J. & Ma, Q. (2008). Aflatoxin B1 degradation by Stenotrophomonas maltophilia and other microbes selected using coumarin medium. International Journal of Molecular Sciences. Vol.9, No.8, pp. 1489-1503, INSS 1422-0067 Nonaka, Y.; Saito, K.; Hanioka, N.; Narimatsu, S. & Kataoka, H. (2009). Determination of aflatoxins in food samples by automated on-line in-tube solid-phase microextraction coupled with liquid chromatography–mass spectrometry. Journal of Chromatography A, Vol.1216, No.20, pp. 4416–4422, ISSN 0021-9673. OBrian, G.R.; Fakhoury, A.M.; Payne, G.A. (2003). Identification of genes differentially expressed during aflatoxin biosynthesis in Aspergillus flavus and Aspergillus parasiticus . Fungal Genetics and Biology, Vol.39, No.2, pp. 118-127, ISSN 1087-1845 Oliveira, T.R.; Barana, A.C.; Jaccoud-Filho, D.S. & Neto, F.F. (2010). Avaliação da contaminação por aflatoxinas totais e zearalenona em variedades de milho crioulo

436

Aflatoxins – Biochemistry and Molecular Biology

(Zea Mays L.) através do método imunoenzimático Elisa. Revista Brasileira de Tecnologia Agroindustrial, Vol.4, No.2, pp. 179-185, ISSN 1981-3686 Park, D.L. (2002). Effect of processing on aflatoxin. Advances in experimental medicine and biology. New York, v. 504, pp. 173-179. Park, M.V.D.Z. & Bos, P.M.J. (2007). Information sources for the detection of emerging mycotoxin risks, Report 320111001, National Institute for Public Health and the Environment, Bilthoven, 2007, 56 pp., Available from http://www.gencat.cat/salut/acsa/320111001.pdf Piermarini, S.; Volpe, G.; Micheli, L.; Moscone, D. & Palleschi, G. (2009). An ELIME-array for detection of aflatoxin B1 in corn samples. Food Control, Vol.20, No.4, pp. 371–375. ISSN 0956-7135 Quinto, M.; Spadaccino, G.; Palermo, C. & Centonze, D. (2009). Determination of aflatoxins in cereal flours by solid-phase microextraction coupled with liquid chromatography and post-column photochemical derivatization-fluorescence detection. Journal of Chromatography A, Vol.1216, No.49, pp. 8636–8641, ISSN 00219673 Ramos, C.R.B.A.; Brasil, E.M.; Geraldine, R.M. (2008). Contaminação por aflatoxinas em híbridos de milho cultivados em três regiões do estado de Goiás. Pesquisa Agropecuária Tropical, Vol.38, No.2, pp. 95-102, ISSN 0278-6915 Reiter, E.; Zentek, J. & Razzazi, E. (2009). Review on sample preparation strategies and methods used for the analysis of aflatoxins in food and feed. Molecular Nutrition & Food Research. Vol.53, No.4, pp. 508-524, ISSN 1613-4133 Reiter, E.; Vouk, F.; Böhma, J. & Razzazi-Fazeli, E. (2010). Aflatoxins in rice – A limited survey of products marketed in Austria. Food Control, Vol.21, No.7, pp. 988–991, ISSN 0956-7135 Ribeiro, J.M.M.; Cavaglieri, J.R.; Fraga, M.E.; Direito, G.M.; Dalcero, A.M. & Rosa, C.A.R. (2006). Influence of Water Activity, Temperature and Time on Mycotoxins Procuction on Barley Rootlets. Letters of Applied Microbiology, Vol.42, No.2, pp. 179184, ISSN 02268254 Romero, A.C.; Ferreira, T.R.B.; Dias, C.T.S; Calori-Domingues, M.A. & Gloria, E.M. (2010). Occurrence of AFM1 in urine samples of a Brazilian population and association with food consumption. Food Control, Vol.21, No.4, pp. 554-558, ISSN 0956-7135 Rubert, J.; Soler, C. & Mañes, J. (2010). Optimization of Matrix Solid-Phase Dispersion method for simultaneous extraction of aflatoxins and OTA in cereals and its application to commercial samples. Talanta, Vol.82, No.1, pp. 567–574, ISSN 00399140 Saha, D.; Acharya, D.; Roy, D.; Shrestha, D. & Dhar, T. (2007). Simultaneous enzyme immunoassay for the screening of aflatoxin B1 and ochratoxin A in chili samples. Analytica Chimica Acta, Vol.584, No.2, pp. 343–349, ISSN 0003-2670 Sassahara, M.; Pontes Neto, D. & Yanaka, E.K. (2005). Aflatoxin occurrence in foodstuff supplied to dairy cattle and aflatoxin M1 in raw milk in the North of Parana State, Food and Chemical Toxicology, Vol.43, No.6, pp. 981-984, ISSN 0278-6915 Sebastia, N.; Soler, C.; Soriano, J. & Mañes, J. (2010) Occurrence of Aflatoxins in Tigernuts and Their Beverages Commercialized in Spain. Journal of Agricultural and Food Chemistry, Vol.58, No.4, pp. 2609–2612, ISSN 0021-8561

Aflatoxins: Contamination, Analysis and Control

437

Sheibani, A. & Ghaziaskar, H. (2009). Pressurized fluid extraction for quantitative recovery of aflatoxins B1 and B2 from pistachio. Food Control, Vol.20, No.2, pp. 124–128, ISSN 0956-7135 Shephard, G. (2009). Aflatoxin analysis at the beginning of the twenty-first century. Analytical and Bioanalytical Chemistry, Vol.395, No.5, pp. 1215–1224, ISSN 1618-2650 Schmidt-Heydt, M.; Abdel-Hadi, A.; Naresh, M. & Geisen, R. (2009). Complex Regulation of the Aflatoxin Biosynthesis Gene Cluster of Aspergillus flavus in Relation to Various Combinations of Water Activity and Temperature. International Journal of Food Microbiology. Vol.135, pp. 231-237, ISSN 0168-1605 Shundo, L.; Almeida, A.; Alaburda, J.; Lamardo, L.; Navas, S.; Ruvieri, V. & Sabino, M. (2009). Aflatoxins and ochratoxin A in Brazilian paprika. Food Control, Vol.20, No.12, pp. 1099–1102, ISSN 0956-7135 Steiner, W.E.; Rieker, R.H. & Battaglia, R. (1988). Aflatoxin contamination in dried figs: Distribution and association with fluorescence. Journal of Agricultural and Food Chemistry, Vol.36, No.1, pp. 88–91, ISSN 0021-8561 Sulyok, M.; Krska, R. & Schuhmacher, R. (2007). A liquid chromatography/tandem mass spectrometric multi-mycotoxin method for the quantification of 87 analytes and its application to semi-quantitative screening of moldy food samples. Analytical and Bioanalytical Chemistry, Vol.389, No.5, pp. 1505–1523. ISSN 1618-2650 Tan, Y.; Chu, X.; Shen, G. & Yu, R. (2009). A signal-amplified electrochemical immunosensor for aflatoxin B1 determination in rice. Analytical Biochemistry, Vol.387, No.1, pp. 82– 86. ISSN 1096-0309 Taylor, M.C.; Jackson, C.J.; Tattersall, D.B.; French, N.; Peat, T.S.; Newman, J.; Briggs, L.J. & Oakeshott, J.G. (2010). Identification and characterization of two families of F420H2 dependent reductases from Mycobacteria that catalyse aflatoxin degradation. Molecular Microbiology. Vol.78, No.3, pp. 561-575, ISSN 1365-2958 Tripathi, S. & Mishra, H.N. (2010). Enzymatic coupled with UV degradation of aflatoxina B1 in red chili powder. Journal of Food Quality. Vol.33, SUPPL. s1, pp. 186-203, ISSN 1745-4557 Trucksess, M.W. & Diaz-Amigo C. (2011). Mycotoxins in Foods Encyclopedia of Environmental Health, 888-897. Ueno, Y. (1986). Trichothecene as environmental toxicants. In: Reviews in Environmental Toxicology 2. Ernest Hodgson (ed). Elsevier, Amsterdan, N.York, Oxford Van der Felx-Klers, H.J.; Dekkers, S.; Kandhai, M. C.; Jeurissen, S.M.F. & Booij, C.J.H. (2010). Indicators for early identification of re-emerging mycotoxin. NJAS- Wageningen Journal of Life Sciences, Vol.57, pp. 133-139, ISSN 1573-5214 Van Egmond, H.P. (1989). Aflatoxin M1: occurrence, toxicity, regulation. In Hans P. van Egmond (Ed.), Mycotoxins in dairy products (pp. 11–55). New York: Elsevier Applied Science. Van-Egmond, H.P.; Schothorst, R.C. & Jonker, M.A. (2007). Regulations Relating to Mycotoxins in Food: Perspectives in a Global and European Context. Analitical and Bioanalytical Chemistry, Vol.389, No.1, pp. 147-157, ISSN 1618-2650 Velázquez, W.R.; Martínez, S.P.; Espinosa, V.H.I.; Vera, M.A.N.; Palacios, E.L. & Rojo, F. (2009). Aflatoxinas totales em raciones de bovinos y AFM 1 em leche cruda obtenida en establos del estado de Jalisco, México. Técnica Pecuaria México, Vol.47, No.2, pp. 223-230, ISSN 0040-1889

438

Aflatoxins – Biochemistry and Molecular Biology

Wagacha, J.M. & Muthomi, J.W. (2008). Mycotoxin problem in Africa: current status, implications to food safety and health and possible management strategies. International Journal of Food Microbioogy . Vol.124, No.1, pp. 1–12, ISSN 0168-1606 Wild, C.P. & Montesano, R. (2009). A model of interaction: Aflatoxins and hepatitis viruses in liver cancer aetiology and prevention. Cancer Letters, Vol.286, No.1, pp. 22-28, ISSN 0304-3835 Wogan, G.N. (1966). Chemical Nature and Biological Effects of the Aflatoxins. Bacteriological Reviews, Baltimore, Vol.30, No.2, pp. 460-470, ISSN 0102-3829 Wogan, G.N.; Paglialunga, S. & Newberne, P. M. (1974) Carcinogenic Effects of Low Dietary Levels of Aflatoxin B1 in Rats. Food and Cosmetics Toxicology, Vol.12, No.5-6, pp. 681685, ISSN 0015-6264 Zachariasova, M.; Cajka, T.; Godula, M.; Malachova, A.; Veprikova, Z. & Hajslova, J. (2010). Analysis of multiple mycotoxins in beer employing (ultra)-high-resolution mass spectrometry. Rapid Communications in Mass Spectrometry, Vol.24, No.22, pp. 33573367, ISSN 0951-4198 Zain, M.E. (2011). Impact of mycotoxins on humans and animals. Journal of Saudi Chemical Society, Vol.15, No.2, pp. 129–144, ISSN 1319-6103 Zinedine, A.; Juan, C.; Soriano, J.; Moltó, J.; Idrissi, L. & Mañes, J. (2007). Limited survey for the occurrence of aflatoxins in cereals and poultry feeds from Rabat, Morocco. International Journal of Food Microbiology, Vol.115, No.1, pp. 124–127, ISSN 0168-1605 Zinedine, A. & Mañes, J. (2009). Occurrence and legislation of mycotoxins in food and feed from Morocco. Food Control, Vol. 20, No 4, pp. 334-344, ISSN 0956-7135. Zorlugenç, B.; Zorlugenç, F.K.; Öztekin, S. & Evliya, I.B. (2008). The influence of gaseous ozone and ozonated water on microbial flora and degradation of aflatoxina B1 in dried figs. Food and Chemical Toxicology. Vol.46, No.12, pp. 3593-3597, ISSN 02786915 Zucchi, T.D.; Moraes, L.A. & Melo, I.S. (2008). Streptomyces sp. ASBV-1 reduces aflatoxin accumulation by Aspergillus parasiticus in peanut grains. Journal of Applied Microbiology, Vol. 105, No.6, (November 2008), pp. 2153–2160, INSS 1364-5072

21 Estimated Daily Intake of Aflatoxin M1 in Thailand Nongluck Ruangwises1, Piyawat Saipan2 and Suthep Ruangwises3 1Department

of Pharmaceutical Chemistry, Faculty of Pharmacy, Mahidol University, Bangkok 2Department of Veterinary Public Health, Faculty of Veterinary Medicine, Khon Kaen University, Khon Kaen 3Department of Veterinary Public Health, Faculty of Veterinary Science, Chulalongkorn University, Bangkok Thailand 1. Introduction Aflatoxins are a group of mycotoxins produced by certain species of Aspergillus. These molds grow on a variety of food and feed commodities and produce aflatoxins under appropriate temperature and humidity (Jay et al., 2005). Aflatoxin B1 (AFB1) is the most potent hepatocarcinogen of this group of mycotoxins. Aflatoxin M1 (AFM1) is a hydroxylated metabolite of AFB1 and is secreted in the milk of mammals that have eaten contaminated foods. AFM1 is also a hepatocarcinogen and is classified in Group 1 (carcinogenic to humans) by the International Agency for Research on Cancer (IARC, 2002). Exposure to AFM1 through milk products is considered to be a serious public health problem. Several countries have established regulatory limits for AFM1 in raw milk and milk products, which vary from country to country. According to the Food and Agriculture Organization of the United Nations (FAO), there are 60 countries that have established regulatory limits for AFM1; the values vary from ND (not detectable) to 15 µg/L (FAO, 2004). The two most prevalent limits are 0.05 µg/L (34 countries) and 0.5 µg/L (22 countries). The European Community has set the maximum permitted level for AFM1 in infant formulae and follow-on formulae, including infant milk and follow-on milk, at 0.025 µg/kg, and in raw milk and heat-treated milk at 0.05 µg/kg (European Commission, 2006). The U.S. regulatory limit for AFM1 is 0.5 µg/L (FAO, 2004). However, several countries, including Thailand, have not yet established regulatory limits for AFM1. The Notification of the Ministry of Public Health No. 265 – the law that regulates the quality of milk products in Thailand – only states that ‘‘…milk products may be contaminated with aflatoxins at a level that is not harmful to human health’’ (Ministry of Public Health, 2003). A national food consumption survey was conducted in Thailand during the years 2002–2004; 18,746 participants were divided into five age groups (Groups 1–5): 0–3, >3–9, >9–19, >19–65, and >65 yr (National Bureau of Agricultural Commodity and Food Standards, 2006). The survey showed that the consumption amounts of four types of milk products – milk powder, school milk, commercial pasteurized milk, and UHT (ultra-high-temperature) milk – comprised approximately 93% of all milk products consumed by the Thai population

440

Aflatoxins – Biochemistry and Molecular Biology

(National Bureau of Agricultural Commodity and Food Standards, 2006). In Thailand, in the morning on every school day, students in Grades 1–6 (age range, 6–12 yr) are served pasteurized milk (200 ml) provided by the School Milk Project (Ruangwises & Ruangwises, 2009). Thailand is administratively divided into 76 provinces; the 45 provinces with the highest population were selected for milk sample collection. The purposes of this study were to investigate whether the concentrations of AFM1 in milk powder, school milk, commercial pasteurized milk, and UHT milk products consumed in Thailand are within the acceptable level for consumption, and to estimate the daily intake of AFM1 for the Thai population.

2. Materials and methods 2.1 Chemicals AFM1 reference standard (from Aspergillus flavus), trifluoroacetic acid, methylene chloride, nhexane, and silica gel 60 were purchased from Sigma-Aldrich (St. Louis, MO, USA). AflaM1TM immunoaffinity columns were purchased from Vicam (Nixa, MO, USA). Spherisorb ODS-2 HPLC columns (5 m, 4.6  250 mm) and C18 Sep-Pak columns were obtained from Waters Corporation (Milford, MA, USA). Solvents (HPLC grade) – acetonitrile, methanol, isopropyl alcohol, and water – were obtained from Merck (Darmstadt, Germany). 2.2 Milk sample collection and sample preparation Milk powder samples were purchased from supermarkets (2 samples/province), while school milk samples were collected from 180 elementary schools (4 schools/province). Commercial pasteurized and UHT milk samples were purchased from supermarkets (2 samples each of pasteurized and UHT milk/province). All milk samples were collected between January 2007 and January 2008. Pasteurized milk samples were frozen at –20 °C until analysis (within one month from the manufacturing date). A total of 450 milk samples were analyzed in this study. Milk powder sample (31.25 g) was reconstituted in 200 ml of distilled water in a 250-ml volumetric flask, mixed well, and adjusted to 250 ml with distilled water (dilution 1: 8). Concentrations of fat, protein, and solid-not-fat (SNF) in reconstituted milk powder samples, analyzed using a MilkoScan 133B (Foss Electric, Hillerød, Denmark), were 4.12 ± 0.36, 3.21 ± 0.08, and 8.59 ± 0.09 g/100 ml (n = 30), respectively. These milk compositions conformed with the Notification of the Ministry of Public Health No. 265, which states that fluid milk with full butter fat must contain fat, protein, and SNF of at least 3.2, 2.8, and 8.25 g/100 ml, respectively (Ministry of Public Health, 2003). The densities of reconstituted and liquid milk samples were determined using 50 ml of milk sample. 2.3 Extraction and determination of Aflatoxin M1 AFM1 was extracted from milk samples using an AflaM1TM immunoaffinity column. The extraction procedure was according to the manufacturer’s recommendations, as previously described by Ruangwises & Ruangwises (2009). In brief, an aliquot of 50 ml of reconstituted milk powder or liquid milk sample was transferred to a 50-ml plastic centrifuge tube and defatted by centrifugation at 3,500 g for 20 min. After the fat was separated, the resulting skimmed milk was transferred into a 50-ml plastic syringe which was attached to an immunoaffinity column. The skimmed milk was allowed to flow into the column by gravity at a rate of 1 ml/min. The column was then washed with 20 ml of water. AFM1 was eluted with 1.25 ml of acetonitrile: methanol (3: 2), followed by 1.25 ml of HPLC water. A total

Estimated Daily Intake of Aflatoxin M1 in Thailand

441

volume of 2.5 ml of eluate was filtered through a nylon filter (0.45 µm) and used for analysis of AFM1 using HPLC. All milk samples were analyzed in duplicate. The complete chromatographic system (Class-LC10; Shimadzu, Kyoto, Japan) consisted of a HPLC pump (model LC-10AD), an auto injector (model SIL-10A), a column oven (model CTO-10A), and a fluorescence detector (model RF-10AXL). The HPLC conditions for analysis of AFM1 were as follows: column – Spherisorb ODS-2; column temperature – 40°C; mobile phase – water: methanol: acetonitrile (57: 23: 20); flow rate – 1 ml/min; detector – fluorescence spectrophotometer (excitation 360 nm; emission 440 nm). The AOAC Official Method 986.16 for detection of aflatoxins M1 and M2 in fluid milk (Cunniff, 1995) was performed to confirm the AFM1 analysis using the immunoaffinity column. In brief, an aliquot of fluid milk or reconstituted milk powder sample (20 ml) was mixed with 20 ml of hot water (80°C), and AFM1 was extracted using a C18 Sep-Pak column. AFM1 was eluted from the column with ether. The ether phase was then cleaned up using a silica gel 60 mini-column; AFM1 was eluted with a mixture of methylene chloride: ethanol (95: 5). The eluate was evaporated to dryness under nitrogen gas; the residue was then dissolved in 0.2 ml of n-hexane. To convert AFM1 to AFM2a, which has a higher extinction coefficient, the solution was derivatized with 0.2 ml of trifluoroacetic acid at 40°C and then evaporated to dryness under nitrogen gas. The residue was dissolved in 2 ml of water: acetonitrile (3: 1). The final solution was filtered through a nylon filter (0.45 µm) and used for analysis of AFM1 by HPLC. The HPLC conditions for analysis of AFM2a were as follows: column – Spherisorb ODS-2 (5 m, 4.6  250 mm); mobile phase – water: acetonitrile: isopropanol (80: 12: 8); flow rate – 1 ml/min; detector – fluorescence spectrophotometer (excitation 365 nm; emission 455 nm). A combination of the two methods was used to confirm the quantification of AFM1 extracted using the AflaM1TM immunoaffinity column. The eluate (2.5 ml) from the immunoaffinity column was evaporated under nitrogen gas to dryness. The residue was dissolved in 0.2 ml of n-hexane and was quantified as described in the AOAC Official Method 986.16, with the addition of 0.2 ml of trifluoroacetic acid. 2.4 Determination of limit of quantification The limit of quantification (LOQ) for AFM1 was determined using the Q2B method of the U.S. Food and Drug Administration (U.S. FDA, 1996). Milk samples (50 ml) were fortified with standard AFM1 at concentrations of 0.0125, 0.025, 0.05, 0.25, and 0.5 µg/L; blank samples were not fortified with standard AFM1. Concentrations of AFM1 in fortified milk samples and blank samples were quantified as described in Extraction and Determintion of AFM1 using the AflaM1TM immunoaffinity column. Calibration curves (n = 12) were obtained by least-square linear regression analysis of the residual peak heights versus fortified AFM1 concentrations. The calculation for LOQ was based on the standard deviation of y-intercepts of linear regression analysis (σ) and the slope (S) using the equation LOQ = 10 σ/S. The LOQ of the method was 0.01 µg/L and the overall recovery across the five concentrations of fortified AFM1 was 85.6%. The precision of the method, expressed as %CV (coefficient of variation), ranged from 2.8 to 5.6%, as previously discussed by Ruangwises & Ruangwises (2009). 2.5 Statistical analysis A randomized block experiment was used to assess the differences between AFM1 concentrations. Duncan’s multiple comparison test was applied to obtain significance levels between the four types of milk samples (P < 0.05). SPSS Statistics version 17.0 for Windows was used for statistical analysis.

442

Aflatoxins – Biochemistry and Molecular Biology

3. Results and discussion Concentrations of AFM1 extracted using an immunoaffinity column with HPLC quantification were comparable to those analyzed using AOAC Official Method 986.16, and also comparable to those obtained from a combination of the two methods (extraction with an immunoaffinity column, derivatization with trifluoroacetic acid, and quantification with HPLC). Comparative results for AFM1 concentrations in liquid milk and milk powder samples obtained from the three procedures are presented in Table 1. In this study, the average density of reconstituted milk powder and liquid milk samples was 1.03 ± 0.027 g/ml (n = 90), which was used for unit conversion. Concentrations and incidence of AFM1 in 450 milk samples are presented in Table 2. Of the 450 samples, 288 (64.0%) were found to be contaminated with AFM1 equal to or above the LOQ of 0.01 µg/L. The incidence of AFM1 in milk powder, school milk, commercial pasteurized milk, and UHT milk samples was 21.1% (19/90), 71.1% (128/180), 78.9% (71/90), and 77.8% (70/90), respectively. Average concentrations of AFM1 found in the four types of milk samples were 0.004 ± 0.009, 0.035 ± 0.028, 0.048 ± 0.034, and 0.045 ± 0.034 µg/L, respectively. In this study, statistical analysis showed that the average concentration of AFM1 in milk powder samples was significant lower than those found in the other three milk products. The average concentration of AFM1 in school milk samples was significantly lower than those found in the commercial pasteurized and UHT milk samples. Of the 19 positive milk powder samples, only 2 samples were contaminated with AFM1 above the EU limit for infant milk products of 0.025 µg/kg. For school milk, commercial pasteurized milk, and UHT milk samples, 68/180 (37.8%), 25/90 (27.8%), and 29/90 (32.2%) samples, respectively, were contaminated with AFM1 within the EU limit of 0.05 µg/kg. Concentrations of AFM1 found in all milk samples were within the U.S. regulatory limit of 0.5 µg/kg. Sample

AFM1 concentration (µg/L) * AOAC Official Method 986.16

Immunoaffinity column + TFA

Immunoaffinity column

Milk Powder (dilution 1:8) Sample A Sample A + 0.05 µg/L AFM1 Sample A + 0.1 µg/L AFM1 Sample A + 0.25 µg/L AFM1 Sample B Sample C Sample D

0.021 0.027 0.119 0.291 0.046 0.031 0.022

0.024 0.028 0.120 0.302 0.047 0.040 0.028

0.026 0.033 0.125 0.299 0.042 0.038 0.023

Liquid Milk Sample 1 Sample 1 + 0.05 µg/L AFM1 Sample 1 + 0.1 µg/L AFM1 Sample 1 + 0.25 µg/L AFM1 Sample 2 Sample 3 Sample 4

0.035 0.079 0.127 0.246 0.062 0.040 0.089

0.032 0.078 0.122 0.247 0.060 0.043 0.085

0.033 0.076 0.129 0.244 0.065 0.039 0.091

All samples were analyzed in duplicate.

*

Table 1. AFM1 concentrations in milk powder and liquid milk samples obtained from three procedures

443

Estimated Daily Intake of Aflatoxin M1 in Thailand

Sample

Samples

Positive (%)

Analyzed

Milk powder Liquid milk Pasteurized school milk Pasteurized commercial UHT commercial Overall

AFM1 concentration (μg/kg) Mean ± SD

Range2

AFM1 incidence1 0.010- 0.050 0.051- 0.075 0.076- 0.100 ≥ 0.101 μg/kg μg/kg μg/kg μg/kg

90

19 (21.1)3 0.004± 0.0094 A5

0.011-0.048

19 (21.1)

-

-

-

180

128 (71.1) 0.035±0.028 B

0.012-0.104

68 (37.8)

50 (27.8)

7 (3.9)

3 (1.7)

90

71 (78.9)

0.048± 0.034 C

0.014-0.109

25 (27.8)

29 (32.2)

14 (15.6)

3 (3.3)

90

70 (77.8)

0.045± 0.034 C

0.012-0.112

29 (32.2)

26 (28.9)

11 (12.2)

4 (4.4)

450

288 (64.0)

0.034± 0.031

0.011-0.112

141 (31.3)

105 (23.3)

32 (7.1)

10 (2.2)

AFM1 incidence of the positive samples Ranges of AFM1 concentrations of the positive samples 3Numbers in parentheses are percentages of each milk product 4Concentrations of AFM1 in reconstituted milk powder samples (1: 8) 5Different letters (A, B, C) denote significant differences between means of each milk product (P < 0.05) 1

2

Table 2. Concentrations and incidence of AFM1 in milk powder and liquid milk samples Since each of the five Thai population groups consumed different types of milk products, calculation of the daily AFM1 intake for each population group was based on the mean AFM1 concentrations in the corresponding milk products. The mean daily AFM1 intake by each group was calculated as follows: mean AFM1 intake (ng AFM1/day) = (mean daily milk intake, kg/day) x (mean AFM1 concentration in the corresponding milk products, µg/kg) x 1,000 (a unit conversion factor, ng/µg). The mean consumption amounts of milk products for the five Thai population groups were 0.429, 0.220, 0.138, 0.059, and 0.036 kg/day, respectively, while the average body weights were 10.1, 20.0, 46.2, 59.4, and 54.5 kg, respectively (National Bureau of Agricultural Commodity and Food Standards, 2006). For Group 1 (age range, 0–3 yr), a mean AFM1 concentration in milk powder samples of 0.004 µg/kg (n = 90) was used for the calculation of daily AFM1 intake. A mean AFM1 concentration of 0.024 µg/kg in milk powder and school milk samples (n = 270) was used for the calculation of daily AFM1 intake for Group 2 (>3–9 yr), while a mean AFM1 concentration of 0.040 µg/kg in school milk, commercial pasteurized milk, and UHT milk samples (n = 360) was used for Group 3 (>9–19 yr). As school milk is consumed by students in Grades 1–6 (6–12 yr), concentrations of AFM1 in school milk samples were used for both Groups 2 and 3. For Group 4 (>19–65 yr) and Group 5 (>65 yr), a mean AFM1 concentration of 0.045 µg/kg in commercial pasteurized and UHT milk samples (n = 180) was used for the calculation of daily AFM1 intake. The estimated daily intakes of AFM1 for the five Thai population groups were 1.63, 5.29, 5.50, 2.63, and 1.62 ng/day, respectively. The estimated daily intakes of AFM1 with respect to body weight for the five population groups were 0.16, 0.26, 0.12, 0.04, and 0.03 ng/kg BW/day, respectively.

444

Aflatoxins – Biochemistry and Molecular Biology

Daily intakes of AFM1 in various regions and countries are presented in Table 3. The Joint FAO/WHO Expert Committee on Food Additives (JECFA) calculated that the daily AFM1 intakes in Africa, the Middle East, Latin America, Europe, and the Far East (using the mean AFM1 concentrations in four different milk products) were 0.002, 0.005, 0.022, 0.023, and 0.36 µg/kg, respectively (JECFA, 2001). The amounts of milk products consumed in the five regions were 0.042, 0.12, 0.16, 0.29, and 0.032 kg/day, respectively. The estimated intakes of AFM1 in the five regions were 0.1, 0.6, 3.5, 6.8, and 12 ng/day, respectively. When AFM1 intakes were calculated with respect to body weight (assuming 60 kg), the estimated daily intakes of AFM1 were 0.002, 0.1, 0.058, 0.11, and 0.20 ng/kg BW/day, respectively (JECFA, 2001). Region/Country

France

Brazil

Spain

Milk consumption1

AFM1 concentration

(kg/day)

(µg/kg)

ng/day

ng/kg BW/day

Africa Middle East Latin America Europe Far East

0.042 0.12 0.16 0.29 0.032

0.002 0.005 0.022 0.023 0.36

0.1 0.6 3.5 6.8 12

0.002 0.1 0.058 0.11 0.20

age 3-14 yr

0.312

0.005-0.052

NA3

0.22

age >15 yr

0.229

0.005-0.05

NA3

0.09

children

0.3604

0.061

23.92

1.045

adults

0.4124

0.031

11.28

0.1885

age 4-9 yr

0.532

0.009696

2.63

0.21

age 10-19 yr age 20-65 yr age >65yr

0.404 0.305 0.407

0.00969 0.00969 0.00969

2.01 1.44 1.94

0.07 0.04 0.05

0.429 0.220 0.138 0.059 0.036 0.176

0.0047 0.0247 0.0407 0.0457 0.0457 0.0320

1.63 5.29 5.50 2.63 1.62 3.33

0.16 0.26 0.12 0.04 0.03 0.12

Thailand age 0-3 yr age >3- 9yr age >9-19 yr age >19-65 yr age >65yr Overall

2

Daily AFM1 intake

Reference

JECFA (2001) JECFA (2001) JECFA (2001) JECFA (2001) JECFA (2001) Leblanc et al. (2005) Shundo et al. (2009) Cano-Sancho et al. (2010)

This study

Average milk consumption for each region or country AFM1 concentrations in five milk products which were used for the calculation of AFM1 intake for each population group (see text for details) 3NA = Data not available 4The values are average milk consumption amounts for both sexes calculated from milk consumption amounts by males and females in each of four population groups presented in the report 5Daily AFM1 intakes were a summation of individual AFM1 intakes from five milk products 6A single value of average AFM1 concentration in three milk products was used for the calculation of daily AFM1 intake for all four Spanish population groups 7Average AFM1 concentrations in corresponding milk products for each Thai population group were used for the calculation of daily AFM1 intakes (see text for explanation) 1 2

Table 3. Daily AFM1 intakes in various regions/countries

Estimated Daily Intake of Aflatoxin M1 in Thailand

445

Leblanc et al. (2005) used individual means of AFM1 concentrations of five milk products – butter (0.05 µg/kg), desserts (0.05 µg/kg), cheeses (0.05 µg/kg), milk (0.005 µg/kg), and ultra-fresh dairy products (0.047 µg/kg) – and daily milk consumption to calculate daily AFM1 intakes for two French population groups: children (3–14 yr) and adults (≥ 15 yr). Daily intakes of AFM1 for each population group were the summation of daily AFM1 intakes from the five milk products; the estimated AFM1 intakes by French children and adults were 0.22 and 0.09 ng/kg BW/day, respectively. Shundo et al. (2009) used AFM1 concentrations in different milk products to estimate daily AFM1 intake by Brazilian children and adults in the city of Sao Paulo. Based on the mean AFM1 concentration in powder milk samples (61 ng/kg) collected from municipal day-care centers and elementary schools, an average milk consumption of 0.412 kg/day, and a body weight of 23 kg, the estimated daily AFM1 intake for children was 1.04 ng/kg BW. For adults, the estimated AFM1 intake was 0.188 ng/kg BW/day, which was calculated using the mean AFM1 concentrations in milk powder and fluid milk samples purchased from supermarkets (31 ng/kg), a daily milk consumption of 0.361 kg, and a body weight of 60 kg. Cano-Sancho et al. (2010) used an average AFM1 concentration of 9.69 ± 2.07 ng/kg found in three milk products (UHT milk, cheese, and yogurt samples) to estimate daily AFM1 intake by four population groups in Catalonia, Spain. Average milk consumption for each of the four population groups – children (4–9 yr), teenagers (10–19 yr), adults (20–65 yr), and elderly (>65 yr) – was 0.532, 0.404, 0.305, and 0.407 kg/day, respectively; while the body weights were 26.2, 54.1, 73.7, and 73.3 kg, respectively. The estimated AFM1 intakes for the four Spanish population groups were 0.21, 0.07, 0.04, and 0.05 ng/kg BW/day, respectively. This study showed that 288 (64.0%) of the 450 milk samples collected from 45 provinces of Thailand were contaminated with AFM1 equal to or more than the LOQ of 0.01 µg/L. Daily intakes of AFM1 in five Thai population groups, calculated using corresponding AFM1 concentrations in milk products consumed by each population group, were comparable to those of other regions and countries. Thai children (3–9 yr) had the highest AFM1 intake, 0.26 ng/kg BW/day, which was comparable to those in the Far East (0.20 ng/kg BW/day), France (3–14 yr; 0.22 ng/kg BW/day), Brazil (adults, 0.188 ng/kg BW/day), and Spain (4–9 yr, 0.21 ng/kg BW/day). Thailand is one of several countries that have not yet established regulatory limits for AFM1 in raw milk and milk products. The present study and our two previous reports (Ruangwises & Ruangwises, 2009, 2010) suggest regular monitoring of raw milk and milk products, and regulatory limits for AFM1 to ensure the quality of raw milk and milk products in Thailand.

4. Acknowledgments This study was financially supported in part by a 2005–2006 Fiscal Research Grant. The authors thank Mrs. Chailai Kuwattananukul and Mr. Sumate Thiangtham for their technical help, and Mr. Christopher Salisbury, Chiang Mai University, Thailand, for reviewing the manuscript.

5. References Cano-Sancho, G., Marin, S., Ramos, A.J., Peris-Vicente, J., & Sanchis, V. (2010). Occurrence of aflatoxin M1 and exposure assessment in Catalonia (Spain). Revista Iberoamericana de Micología, Vol. 27, No. 3, (July-September 2010), pp. 130-135, ISSN 1130-1406

446

Aflatoxins – Biochemistry and Molecular Biology

Cunniff, P.A. (Ed.). (1995). Official Methods of Analysis (16th edition), AOAC International, ISBN 0-935584-54-4, Gaithersburg, Maryland [Section: AOAC Official Method 986.16 – Aflatoxins M1 and M2 in fluid milk] European Commission (2006). Commission Regulation (EC) No. 1881/2006 of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs. Official Journal of the European Union, 20.12.2006, pp. L 364/5 – L 364/24, ISSN 1725-2555 FAO (Food and Agriculture Organization of the United Nations) (2004). Worldwide Regulations for Mycotoxins in Food and Feed in 2003, Food and Nutrition Paper 81, FAO, ISBN 92-5-105162-3, Rome IARC (International Agency for Research on Cancer) (2002). IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, Vol. 82: Some Traditional Herbal Medicines, Some Mycotoxins, Naphthalene and Styrene, World Health Organization (WHO), IARC Press, ISBN 92-832-1282-7, Lyon, France Jay, J.M., Loessner, M.J., & Golden, D.A. (2005). Modern Food Microbiology (7th edition), Springer Science and Business Media, ISBN 0-387-23180-3, New York JECFA (Joint FAO/WHO Expert Committee on Food Additives) (2001). Safety Evaluation of Certain Mycotoxins in Food: Aflatoxin M1, WHO Food Additives Series No. 47, ISBN 92-4-120940-2, Geneva, Switzerland Leblanc, J.C., Tard, A., Volatier, J.L., & Verger, P. (2005). Estimated dietary exposure to principal food mycotoxins from the First French Total Diet Study. Food Additives and Contaminants, Part A, Vol. 22, No. 7, (July 2005), pp. 652–672, ISSN 1944-0049 Ministry of Public Health (2003). Notification of the Ministry of Public Health No. 265 (B.E. 2545): Cow’s milk. The Royal Thai Government Gazette, Vol. 120, Special Issue 4D, (January 2003), Bangkok, Thailand (In Thai) National Bureau of Agricultural Commodity and Food Standards (2006). Food Consumption Data of Thailand, Ministry of Agriculture and Co-operatives, ISBN 974-403-423-8, Bangkok, Thailand (In Thai) Ruangwises, S., & Ruangwises, N. (2009). Occurrence of aflatoxin M1 in pasteurized milk of the School Milk Project in Thailand. Journal of Food Protection, Vol. 72, No. 8, (August 2009), pp. 1761–1763, ISSN 0362-028X Ruangwises, N., & Ruangwises, S. (2010). Aflatoxin M1 contamination in raw milk within the central region of Thailand. Bulletin of Environmental Contamination and Toxicology, Vol. 85, No. 2, (August 2010), pp. 195–198, ISSN 0007-4861 Shundo, L., Navas, S.A., Lamardo, L.C.A., Ruvieri V., & Sabino, M. (2009). Estimate of aflatoxin M1 exposure in milk and occurrence in Brazil. Food Control, Vol. 20, No. 7, (July 2009), pp. 655–657, ISSN 0956-7135 U.S. FDA (United States Food and Drug Administration) (1996). Guidance for Industry – Q2B Validation of Analytical Procedures: Methodology, Department of Health and Human Services, Center for Drug Evaluation and Research, Rockville, Maryland, Available from: http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatory Information/Guidances/ucm073384. pdf

22 Influence of Soluble Feed Proteins and Clay Additive Charge Density on Aflatoxin Binding in Ingested Feeds William F. Jaynes and Richard E. Zartman Texas Tech University USA 1. Introduction Hartley et al. (1963) isolated and identified toxic metabolites of Aspergillus flavus as aflatoxins B1, B2, G1, and G2; named from the blue and green fluorescence of the compounds under ultraviolet light. The aflatoxins are a group of mycotoxins produced by Aspergillus fungi that are both toxic and carcinogenic to animals and humans (Murphy et al., 2006). Aflatoxin B1 (AfB1) and mixtures of B1, G1, and M1 are proven human carcinogens (IARC, 1993). Aflatoxin B1 is the most toxic (Figure 1), most abundant, and the most potent natural carcinogen known (Squire, 1981). An estimated 4.5 billion people living in developing countries are chronically exposed to uncontrolled amounts of aflatoxins (Williams et al., 2004). Iraq produced aflatoxins for use in biological warfare between 1985 and 1991, but the weapons had little military value (Zilinskas, 1997). After ingestion, aflatoxins are converted to the reactive 8,9-epoxide form that can bind to DNA and proteins. Aflatoxin consumption results in diseases that are loosely called aflatoxicoses. Chemically, aflatoxins are derivatives of difuranocoumarin (Bennett & Klich, 2003). Various methods have been used to reduce the toxicity of aflatoxin-contaminated grains. Cleaning to remove damaged corn kernels is sometimes effective in reducing aflatoxin concentrations, but undamaged kernels can also contain high aflatoxin concentrations (Vincelli et al., 1995). Treatment with anhydrous ammonia can be used to detoxify grain that is to be used on the farm (Vincelli et al., 1995). Brekke et al. (1977) ammoniated trout feed contaminated with 180 µg/kg aflatoxins, which inactivated the aflatoxins and reduced the carcinogenicity to a level not significantly different than the control. Grove et al. (1981) examined the ammoniation products of aflatoxin model coumarins and determined that the keto group in the cyclopentene ring is required for ammonia-induced decomposition. Nixtamalization is an Aztec word that means lime-cooked corn (Herrera et al., 1986) and is an ancient method used to soften grain before it is used in foods. Nixtamalization also increases protein quality and niacin bioavailability (Sefa-Dedah et al., 2004). The strong alkalinity imparted by lime (CaO, Ca(OH)2 ) might have a similar effect on aflatoxins as ammonia. Arrriola et al., (1988) examined the effect of nixtamalization on aflatoxin fate during tortilla preparation using 2-10% CaO. Nixtamalization decreased aflatoxin concentrations at even the lowest CaO concentrations. However, nixtamalization did not

448

Aflatoxins – Biochemistry and Molecular Biology

reduce the 1360-1896 µg/kg initial aflatoxin concentrations down to the allowable value of 20 µg/kg. Ammoniation and other detoxifying methods, however, are not approved or sanctioned by the U.S. Food and Drug Administration (Sweets & Wrather, 2009). Clays are colloidal or near-colloidal hydrous aluminum silicates that are more or less plastic when moist (Bates, 1969). Bentonites are natural materials that dominantly consist of clay minerals in the smectite group (Hosterman & Patterson, 1992). Smectites are a group of phyllosilicate minerals that include montmorillonite, beidellite, nontronite, saponite, and hectorite (Odom, 1984). Bentonite was the name given by Wilbur G. Knight in 1898 to deposits in the Benton Shale near Rock River, Wyoming (Hosterman & Pattterson, 1992). Bentonite deposits contain altered volcanic ash glass shards and field evidence suggests that bentonites formed from ash that fell into shallow lakes or seas (Bates, 1969). Smectites, vermiculites, talc, and pyrophyllite are structurally-related 2:1 clay minerals, but talc and pyrophyllite have zero layer charge and do not expand in water. Smectites and vermiculites characteristically expand in water along the crystallographic c-axis to form an interlayer region. Structurally, the 2:1 clay minerals consist of an octahedral aluminum or magnesium oxide sheet sandwiched between two tetrahedral silica sheets. Unlike talc and pyrophyllite, smectites and vermiculites have isomorphic chemical substitutions of Al3+ for Si4+ (tetrahedral charge) and Mg2+ for Al3+ (octahedral charge) that impart a negative charge to the mineral surface and a cation exchange capacity (CEC). Inorganic exchange cations, such as Na+ and Ca2+, compensate for the negative charge on smectite and vermiculite surfaces. Smectite CECs range from 50 to 129 cmol/kg and vermiculite CECs range from 130 to 210 cmol/kg (Mermut & Lagaly, 2001; van Olphen & Fripiat, 1979). Bentonites are relatively pure, commercial deposits of smectites found throughout the world that can be mined, but smectites, vermiculites, and other clay minerals commonly also occur in soils and sedimentary deposits. In soils, clays retain exchangeable cations, such as Ca2+, Mg2+, and NH4+, which are essential plant nutrients. Vermiculite expansion (Ca-saturated) in water is limited to ~1.5 nm, but smectites (Ca-saturated) expand to 1.9 nm or more (McEwan & Wilson, 1984). Free expansion of smectites in water is almost unlimited. Completely dispersed smectite particles consist of single unit cells with no c-axis direction repeat distance (Eberl et al., 1998). Interlayer expansion of air-dried (32% relative humidity) samples of Na-smectite and Ca-smectite is illustrated in Figure 1 with characteristic basal spacings after McEwan & Wilson (1984). The single water layer and Na+ cations in Na-smectite interlayers (Figure 1) is ~0.25 nm and the water bilayer and Ca2+ cations in Ca-smectite interlayers is ~0.52 nm. Calcium- and magnesium-saturated smectites yield similar basal spacings. The replacement of inorganic exchange cations in smectites and vermiculites with organic cations can result in interlayer expansion. Jaynes & Boyd (1991) exchanged the organic cation, hexadecyltrimethylammonium (HDTMA), for the inorganic cations in a lowcharge smectite, a high-charge smectite, and a vermiculite, which produced expanded interlayer basal spacings of 1.8, 2.3, and 2.8 nm, respectively. Polymer adsorption to clays can also produce interlayer expansion. Polyvinylpyrrolidone (PVP) expanded SAz highcharge montmorillonite to ~2.3 nm (Blum & Eberl, 2004). Smectite layer charge ranges from 0.2 to 0.6 per O10(OH)4 unit , whereas, vermiculite layer charge ranges from 0.6 to 0.9 (Bailey, 1980). Vermiculites are hydrous minerals that form by the weathering of micas and have a platey mica-like morphology (Newman & Brown, 1987). The name “vermiculite” is more commonly used for macroscopic heat-expanded (800 – 1100 °C) vermiculite particles that are used as a packing material, plant media, insulation, and

Influence of Soluble Feed Proteins and Clay Additive Charge Density on Aflatoxin Binding in Ingested Feeds

449

construction material. This heated vermiculite should not be be confused with the natural mineral because heat-treatment greatly alters the properties. Sepiolite, palygorskite, and the zeolites are structurally much different than smectites and vermiculites and do not have interlayers. Sepiolite and palygorskite are fibrous, non-expandable, hydrous magnesium aluminosilicates. There are some health concerns about the possible effects of inhaled fibrous minerals. Bellman et al. (1997) used intratracheal instillation studies in rats to evaluate the carcinogenic potential of sepiolites. A short-fiber sepiolite from Spain showed no evidence of carcingenic potential, but a long-fiber sepiolite from China had a more pronounced fibrotic response (Bell et al., 1997). Sepiolite (CEC = 20–40 cmol/kg) and palygorskite (CEC = 5-30 cmol/kg) contain internal channels with exchangeable cations and water (Singer, 2002). Zeolites (e.g. clinoptilolite, erionite, analcime, mordenite) are framework-structure, three-dimensional aluminosilicate minerals with interconnected channels and cages that contain exchangeable cations (CEC = 220-570 cmol/kg) and adsorbed water (Boettinger & Ming, 2002). The internal channels in sepiolite (0.37 x 1.06 nm), palygorskite (0.37 x 0.64 nm), and zeolites (0.26 x 0.26 to 0.74 x 0.74 nm) are too small to accomodate aflatoxins. Hence, aflatoxins can only adsorb to external sites on these minerals. Commercial clay additives have been used to prevent caking and improve the physical properties of animal feeds. The decreased toxicity of aflatoxins observed for contaminated animal feed mixed with clay feed additives has stimulated research on clay additives to prevent mycotoxicosis. The commercial clay feed additives, Novasil, Novasil plus, AstraBen 20, and Astra-Ben 20A, are bentonites that primarily consist of the smectite group mineral, montmorillonite. Animal feeding studies have demonstrated that Novasil, Novasil plus, Astra-Ben 20, Astra-Ben 20A, Na-bentonite, zeolite, and sepiolite feed additives can effectively reduce or prevent the toxicity caused by feed contaminated with Aspergillus mycotoxins, such as AfB1 (Phillips et al., 1988, 1995; Scheideler 1993; Schell et al., 1993a, 1993b; Edrington et al., 1996; Abdel-Wahhab et al., 1999; Miazzo et al., 2000; Diaz et al., 2004; Pimpukdee et al. 2004; Bailey et al., 2006; Fairchild et al., 2008; Magnoli et al., 2008). Ruminant animals, such as cattle and sheep, can tolerate higher aflatoxin levels and longer low-level intake periods than simple-stomached animals (Vincelli et al., 1995). The adsorption of aflatoxins to ingested soil minerals might partly explain the greater aflatoxin tolerance of ruminants. Soil ingested by cattle averaged 14% of the dry weight of fecal matter and increased as forage availability decreased (Mayland et al., 1975). Soil ingestion by grazing sheep in March exceeded 30% of dry matter intake at 2 of the 11 sites in midWales (Abrahams & Steigmajer, 2003). Winfree and Allred (1992) measured significant aflatoxin adsorption to bentonite from methanol/water, which is commonly used in the extraction and measurement of aflatoxins in contaminated feed. Gallo et al. (2010) developed a more aggressive extraction procedure using acetone rather than methanol to more accurately measure aflatoxins in feeds that contain feed additives. Deng et al. (2010) measured smectite interlayer expansion of >1.2 nm that was stable to 400 °C after AfB1 treatment, which demonstrated that AfB1 adsorbs to interlayer clay surfaces. Interlayer clay surfaces account for most of the ~800 m2/g surface area of smectites, such as montmorillonite. From infrared spectroscopy, Deng et al. (2010) concluded that hydrogen bonds between AfB1 carbonyl groups and the hydration water of exchangeable cations in clays is the dominant bonding force under humid conditions. Aflatoxin adsorption from aqueous corn and peanut meal to feed additives was consistent with animal feeding studies

450

Aflatoxins – Biochemistry and Molecular Biology

that used the feed additives, Novasil, Novasil Plus, Astra-Ben 20, Astra-Ben 20A, sepiolite, and activated (Norit-A) carbon (Jaynes et al., 2007; Seifert et al., 2010). Feed additives that effectively reduced or prevented aflatoxin toxicity in feeding studies adsorbed more AfB1 from aqueous corn and peanut meal. Animal feedings studies (in vivo) are the surest way to identify effective feed additives, but are much too expensive for routine use. Hence, various approaches have been used for the in vitro evaluation of potential feed additives. Feed additive in vitro test methods should produce results that are consistent with animal feeding studies. Unfortunately, most animal feeding studies evaluated only one or a few feed additives, which makes comparisons of relative effectiveness difficult. This also frustrates efforts to identify feed additive properties related to effective aflatoxin toxicity reduction. In contrast, Schell et al. (1992a) evaluated a sodium calcium bentonite (Novasil), a calcium bentonite (Astra-Ben 20), a sodium bentonite (FD-181), a zeolite (Zeobrite), a palygorskite (Min-U-Gel), and a sepiolite (Sepiolgel UF). Phillips et al. (1988) measured AfB1 adsorption from water to aluminas, zeolites, silicas, phyllosilicates, a Mn-exchanged phyllosilicate, and an acid-activated phyllosilicate. Winfree & Allred (1992) measured a 70% reduction in AfB1 concentrations in methanol/water extracts of trout feed 1 hour after 10% bentonite was added to moistened feed. Grant & Phillips (1998) fitted AfB1 adsorption from water to Novasil data to the Langmuir and other isotherm equations to calculate adsorption capacities. Jaynes et al. (2007) measured AfB1 adsorption from aqueous corn meal after extraction with 60% methanol. Siefert et al. (2010) measured AfB1 adsorption to clays in aqueous peanut meal and total extractable aflatoxins in peanut meal/clay water extracts. Vekiru et al. (2007) measured AfB1 adsorption to bentonites and charcoal from acetate buffer, artificial gastric fluid, and from gastric fluid. Thieu and Pettersson (2008) measured AfB1 adsorption to zeolite and bentonite in simulated gastrointestinal fluids. Dixon et al. (2008) used X-ray diffraction, infrared spectroscopy, cation exchange capacity, and particle size to measure smectite purity and the Langmuir isotherm to measure aflatoxin adsorption from water. To identify feed additives that can effectively bind aflatoxins in ingested feed, in vitro tests should model the environment of ingested feed/feed additive as accurately as practical. Proteins are soluble ionic polymers of amino acids that can adsorb to clay surfaces. Living organisms, animal feed, and human food contain proteins. Lipson & Stotzky (1984) showed that the proteins chymotrypsin, ovalalbumin, and lysozyme adsorbed to montmorillonite and reduced adsorption of the Reovirus. Perez-Castells et al. (1985) puried collagen protein from calf skin and adsorbed 0.4 mg of collagen to 1 mg of sepiolite. Similarly, Garwood adsorbed 97 g of the enzymatic protein, glucose oxidase, to 100 g of Na-montmorillonite. Ralla et al. (2010) used a smectite clay to adsorb and separate proteins. Aflatoxin-contaminated peanuts are unsuitable as food or feed and are used to produce peanut oil (Siefert et al., 2010) and defatted peanut meal is a byproduct of peanut oil production. Siefert et al. (2010) used Astra-Ben 20A to bind aflatoxins in defatted peanut meal in the insoluble residue and produce an aflatoxin-free water-soluble protein extract. Addition of 0.2% Astra-Ben 20A to peanut meal decreased total aflatoxins in the soluble protein extracts from 50 to 4.8 µg/kg. The addition of 2% Astra-Ben 20A decreased total aflatoxins in the soluble fraction to 0 µg/kg, but decreased protein recovery by 37%. Soluble protein adsorption to clay additives in ingested feed might adsorb to clay feed additives and block aflatoxin adsorption. Other soluble compounds in feed, such as polysaccharides, might also adsorb to clay feed additives and block potential aflatoxin binding sites. Chenu et al. (1985) adsorbed ~400 mg of the polysaccharide, scleroglucan, to 1 g of Na-montmorillonite.

Influence of Soluble Feed Proteins and Clay Additive Charge Density on Aflatoxin Binding in Ingested Feeds

451

Fig. 1. Chemical structure of aflatoxin B1 and illustration of interlayer expansion of air-dried calcium-saturated and sodium-saturated smectites.

452

Aflatoxins – Biochemistry and Molecular Biology

Decker (1980) measured the adsorption of 1 mg AfB1 to 100 mg of activated carbon (Norit-A) from aqueous media at pH 7 and suggested activated carbon might be used to prevent animal and human absorption of aflatoxins from contaminated foodstuffs. Early animal feeding studies by Hatch et al. (1982), Dalvi & Ademoyero (1984), and Dalvi & McGowan (1984) indicated that activated carbon can reduce aflatoxicosis. Similarly, bentonites and activated carbon reduced excretion of aflatoxin M1 in milk cows, turkey poults, and goats in feeding studies by Veldman (1992), Edrington et al. (1996), and Rao & Chopra (2001). However, animal feeding studies by Kubena et al. (1990), Bonna et al. (1991), Edrington et al. (1996), and Diaz et al. (2004) concluded that activated carbon does not effectively reduce aflatoxin toxicity to fed animals or is not as effective as clay additives. Diaz & Smith (2005) did not recommend routine inclusion of activated charcoal in diets after reviewing activated charcoal use in animal feeding studies. Vekiru et al. (2007) measured AfB1 binding by various sorbents and identified another drawback to activated carbon use as a feed additive; unlike the bentonites, activated carbon adsorbed the essential nutrients, vitamin B12 and biotin. Clay feed additives are marketed and sold in the United States as anti-caking agents to improve the physical properties of feed because U.S. Food and Drug Administration (FDA) regulations do not permit feed additive companies to claim that feed additives can bind mycotoxins and reduce mycotoxicoses. Therefore, feed additive companies have little financial incentive to develop additives with improved mycotoxin binding. Feed additives are mixed with dry feeds and, hence, mycotoxin binding to clays must occur after ingestion. During digestion, pH, feed composition, and other factors can affect mycotoxin binding to feed additives. Mycotoxin adsorption to feed additives can sequester the toxins and limit absorption by animals or humans. However, feed additives that effectively remove mycotoxins from water might not prevent toxicity to animals from contaminated feed because the adsorption of soluble feed or digestive compounds might block mycotoxin adsorption to feed additives. In this study, the effects of soluble feed compounds and clay layer charge on the adsorption of AfB1 to commercial feed additive clays, reference clays, and activated carbon will be examined. Adsorption of AfB1 to a variety of commercial feed additives and reference clays were measured from water and aqueous corn meal. Clays and activated carbon were treated with aqueous corn and peanut meal extracts to simulate the adsorption of soluble feed compounds to feed additives ingested with feed. The physical and chemical properties of materials treated with corn- and peanut-meal water extracts will be measured and related to aflatoxin adsorption. The adsorption of AfB1 to clays with a wide range in layer charge will be measured to determine layer charge effects on AfB1 adsorption.

2. Materials and methods The reference clay samples, SWy-2 (SWy), SAz-1 (SAz), SepSp-1 (SepSp), and SHCa-1 (SHCa) were obtained from the Source Clay Repository of the Clay Minerals Society located at Purdue University (West Lafayette, IN). The SWy sample is a low-charge sodium montmorillonite from Wyoming and SAz is a high-charge calcium montmorillonite from Arizona. The SepSep sample is a sepiolite from Spain and SHCa is a hectorite from California. Sepiolite is a fibrous magnesium silicate clay mineral and hectorite is a lowcharge magnesium layer silicate. Both montmorillonite and hectorite are expanding smectite-group minerals. Novasil and Novasil plus (low-charge montmorillonite) are products of Trouw Nutrition, which is a division of Englehard Corporation, Chemical

Influence of Soluble Feed Proteins and Clay Additive Charge Density on Aflatoxin Binding in Ingested Feeds

453

Catalysts Group (600 East McDowell Road, Jackson, MS). A vermiculite sample, VSC, from a mine in South Carolina was obtained from the W.R. Grace Company. Vermiculites have a higher CEC/layer charge than smectites. Clay mineral samples were Na-saturated by treatment with NaCl, 0.35Li-SAz > 0.50Li-SAz.

464

Aflatoxins – Biochemistry and Molecular Biology

4. Conclusions The relative amounts of aflatoxin B1 retained from aqueous corn meal by clays and activated carbon after 60% methanol extraction were consistent with animal feeding study results, but animal feeding study results were not consistent with adsorption from water. Aflatoxin retention from aqueous feed seems to model aflatoxin adsorption to ingested feed additives more effectively than simple adsorption from water. Clay and activated carbon samples that were treated with aqueous extracts of corn and peanut meal had increased C and N contents and decreased surface areas. Smectite interlayer basal spacing was increased after treatment with the peanut extracts. The adsorption of bovine serum albumin protein and aflatoxin B1 also decreased after corn/peanut meal extract treatment. These data indicate that soluble compounds in corn and peanut meal extracts adsorbed to clay and activated carbon surfaces and adversely affected aflatoxin binding. Low-charge smectites, such as Novasil plus and reduced-charge SAz, retained more aflatoxin B1 from aqueous corn meal than higher charge minerals, such as vermiculite (VSC) and high-charge smectite (SAz). The selection of lowcharge smectite feed additives might assure greater aflatoxin binding and toxicity reduction. The use of in vitro aflatoxin adsorption tests from aqueous feed might be used to identify feed additives that can effectively bind aflatoxins in ingested feed. The correlation between animal feeding study results and in vitro aflatoxin adsorption from feed tests might be improved by use of gastrointestinal fluids or other factors to make the chemical environment of feed additives in adsorption tests more like ingested toxins. However, adsorption of soluble feed compounds to feed additives appears to have a strongly adverse effect on aflatoxin binding to ingested feed additives.

5. References Abdel-Wahhab, M.A., Nada, S.A., & Amra, H.A. 1999. Effect of aluminosilicates and bentonite on aflatoxin-induced developmental toxicity in rat. J. Appl. Toxicol. 19, 199-204. Abrahams, P.W., & Steigmajer, J. 2003. Soil ingestion by sheep grazing the metal enriched floodplain soils of mid-Wales. Environ. Geochem. Health 25, 17-24. AOCS, 1999a. Aflatoxin standards. Official method Aj 0-88. Sampling and Analysis of Vegetable Oil Source Materials. American Oil Chemists Society. AOCS, 1999b. Total aflatoxins (B1, B2, and G1) in corn, cottonseed, peanuts, and peanut butter. Official method Aj 6-95. Sampling and Analysis of Vegetable Oil Source Materials. American Oil Chemists Society. Arriola, M.C., Porres, E., Cabrera, S., Zepeda, M., & Rolz, C. 1988. Aflatoxin fate during alkaline cooking of corn for tortilla preparation. J. Agric. Food Chem. 36, 530-533. Asis, R., Di Paola, R.D., & Aldao, M.A.J. 2002. Determination of aflatoxin B1 in highly contaminated peanut samples using HPLC and ELISA. Food and Agricultural Immunology 14, 201-208. Bailey, C.A., Latimer,G.W., Barr, A.C., Wigle, W.L., Haq, A.U., Balthrop, J.E. & Kubena, L.F. 2006. Efficacy of montmorillonite clay (Novasil plus) for protecting full-term broilers from aflatoxicosis. J. Appl. Poult. Res. 15, 198-206. Bailey, S.W. 1980. Summary of recommendations of AIPEA nomenclature committee. Clays Clay Miner. 28, 73-78. Bates, R.L. 1969. Clay. pp. 117-155. In: Geology of The Industrial Rocks and Minerals. Dover, New York.

Influence of Soluble Feed Proteins and Clay Additive Charge Density on Aflatoxin Binding in Ingested Feeds

465

Bell, B., Muhle, H., & Ernst, H. Investigations on health-related properties of two sepiolite samples. Environ. Health Perspect. 105 (Suppl 5) 1049-1052. Bennett, J.W., & Klich, M. 2003. Mycotoxins. Clinical Microbiology Reviews 16, 497-516. Blum, A.E., & Eberl, D.D. 2004. Measurement of clay surface areas by polyvinylpyrrolidone (PVP) sorption and its use for quantifying illite and smectite abundance. Clays Clay Miner. 52, 589-602. Boettinger, J.L., & Ming, D.W. 2002. Zeolites. pp. 585-610. In Dixon, J.B., & Schulze, D.G. (eds.) Soil Mineralogy with Environmental Applications. SSSA Book Series, no. 7. Soil Science Society of America, Madison, WI. Bonna, R.J., Aulerich, R.J., Bursian, S.J., Poppenga, R.H., Braselton, W.E., & Watson, G.L. 1991. Efficacy of hydrated sodium aluminosilicate and activated charcoal in reducing the toxicity of dietary aflatoxin to mink. Arch. Environ. Contam. Toxicol. 20, 441-447. Bradford, M.M. 1976. A refined and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248-254. Brekke, O.L., Sinnhuber, R.O., Peplinski, A.J., Wales, J.H., Putnam, G.B., Lee, D.J., & Ciegler, A. 1977. Aflatoxin in corn: ammonia inactivation and bioassay with rainbow trout. Appl. Environ. Microbiol. 34, 34-37. Carter, D.L., Mortland, M.M., & Kemper, W.D. 1986. Specific surface. pp. 413-423 In: Methods of Soil Analysis: Part I. Physical and Mineralogical Methods, 2nd edtiion (A. Klute, editor). Soil Science Society of America, Madison, WI. Chenu, C., Pons, C.H., & Robert, M. 1985. Interaction of kaolinite and montmorillonite with neutral polysaccharides. In: Schultz, L.G., van Olphen, H., & Mumpton, F.A. (eds.) Proceedings of the International Clay Conference 1985, Denver, CO. pp. 375-381, The Clay Minerals Society, Bloomington, IN. Dalvi, R.R. & Ademoyero, A.A. 1984. Toxic effect of aflatoxin B1 in chickens given feed contaminated with Aspergillus flavus and reduction of the toxicity by activated charcoal and some chemical agents. Avin Dis. 28, 61-69. Dalvi, R.R. & McGowan, C. 1984. Experimental induction of chronic aflatoxicosis in chickens by purified aflatoxin B1 and its reversal by activated charcoal, phenobarbitol and reduced glutathione. Poult. Sci. 63, 485-491. Decker, W.J. 1980. Activated charcoal adsorbs aflatoxin B1. Vet. Hum. Toxicol. 22, 388-389. Deng, Y., Barrientos Velazquez, A.L., Billes, F., & Dixon, J.B. 2010. Bonding mechanisms between aflatoxin B1 and smectite. Appl. Clay Sci. 50, 92-98. Diaz, D.E. & Smith, T.K. 2005. Mycotoxin sequestering agents: Practical tools for the neutralisation of mycotoxins. In Diaz, D.E. (editor) The Mycotoxin Blue Book. pp 323340. Nottingham University Press. Diaz, D.E., Hagler, W.M., Blackwelder, J.T., Eve, J.A., Hopkins, B.A., Anderson, K.L., Jones, F.T., & Whitlow, L.W. 2004. Aflatoxin binders II: reduction of aflatoxin M1 in milk by sequestering agents of cows consuming aflatoxin in feed. Mycopathologia, 157, 233-241. Dixon, J.B., Kannewischer, Tenorio Arvide, M.G., & Barrientos Velazquez, A.L. 2008. Aflatoxin sequestration in animal feeds by quality-labeled smectite clays: an introductory plan. Appl. Clay Sci. 40, 201-208. Eberl, D.D., Nüesch, R, Ŝuchá, V., & Tsipursky, S. 1998. Measurement of fundamental illite particle thicknesses by X-ray diffraction using PVP-10 intercalation. Clays Clay Miner. 46, 89-97.

466

Aflatoxins – Biochemistry and Molecular Biology

Edrington, T.S., Sarr, A.B., Kubena, L.F., Harvey, R.B., & Phillips, T.D. 1996. Hydrated sodium calcium aluminosilicate (HSCAS), acidic HSCAS, and activated charcoal reduce urinary excretion of aflatoxin M1 in turkey poults. Lack of effect by activated charcoal on aflatoxicosis. Toxicol. Lett. 89, 115-122. Fairchild, A.S., Croom, J., Grimes, J.L., & Hagler, W.M. 2008. Effect of Astra-Ben 20A on broiler chicks exposed to aflatoxin B1 or T2 toxin. Int. J. Poult. Sci. 7, 1147-1151. Gallo, A., Masoero, F., Bertuzzi, T., Piva, G., & Pietra, A. 2010. Effect of the inclusion of adsorbents on aflatoxin B1 quantification in animal feedstuffs. Food Additives and Contaminants 27, 54-63. Garwood, G.A., Mortland, M.M., & Pinnavaia, T.J. 1983. Immobilization of glucose oxidase on montmorillonite clay: hydrophobic and ionic modes of binding. Journal of Molecular Catalysis, 22, 153-163. Grant, P.G. & Phillips, T.D. 1998. Isothermal adsorption of aflatoxin B1 on HSCAS clay. J. Agric. Food Chem. 46, 599-605. Grove, M.D., Plattner, R.D., & Weisleder, D. 1981. Ammoniation products of an aflatoxin model coumarin. J. Agric. Food Chem. 29,1161-1164. Hartley, R.D., Nesbitt, B.F., & O’Kelly, J. 1963. Toxic metabolites of Aspergillus flavus. Nature 198,1056-1058. Hatch, R.C., Clark, J.D., Jain, A.V., & Weiss, R. 1982. Induced acute aflatoxicosis in goats: treatment with activated charcoal or dual combinations of oxytetracycline, stanozol and activated charcoal. Am. J. Vet. Res. 43, 644-648. Herrera, M., Zyman, J., Pena, J.G., Segurajauregui, J.S., & Vernon, J. 1986. Kinetic studies on the alkaline treatment of corn (Zea mays) for tortilla preparation. J. Food Sci. 51, 1486-1490. Hiemenz, P.C. 1986. Principles of Colloid and Surface Chemistry. Marcel Dekker, New York, 398-407. Hofmann, U. & Klemen, R. 1950. Verlust der Austauschfahigkeit von Lithiumen an Bentonit durch Erhitzung: Z. Anorg. Allg. Chem. 262, 95-99. Hosterman, J.W., & Patterson, S.H. 1992. Bentonite and fuller’s earth resources of the United States: U.S. Geological Survey Professional Paper 1522. U.S. Government Printing Office. IARC. 1993. Aflatoxins: Naturally occurring aflatoxins (Group 1), aflatoxins M1 . Int. Agency Res. Cancer 56, 245. Jaynes, W.F. & Bigham, J.M. 1987. Charge reduction, octahedral charge, and lithium retention in heated, Li-saturated smectites. Clays Clay Miner. 35, 440-448. Jaynes, W.F., & Boyd, S.A. 1991. Clay mineral type and organic compound sorption by hexadecyltrimethylammonium-exchanged clays. Soil Sci. Soc. Am. J. 55, 43-48. Jaynes, W.F., Zartman, R.E., & Hudnall, W.H. 2007. Aflatoxin B1 adsorption by clays from water and corn meal. Appl. Clay Sci. 36, 197-205. Jaynes, W.F., Zartman, R.E., Green, C.J., San Francisco, M.J., & Zak, J.C. 2005. Castor toxin adsorption to clay minerals. Clays Clay Miner. 53, 268-277. Katzin, B.J., Collins, E.J., & Robertus, J.D. 1991. The structure of ricin A chain at 2.5 Å. Proteins: Structure, Function, and Genetics, 10, 251-259. Kubena, L.F., Harvey, R.B., Phillips, T.D., Corrier, D.E., & Huff, W.E. 1990. Diminution of aflatoxicosis in growing chickens by the dietary addition of hydrated sodium calcium aluminosilicate. Poult. Sci. 69, 727-735. Lagaly, G. & Weiss, A. 1976. The layer charge of smectitic layer silicates: In Proc. Int. Clay Conf., Mexico City, 1975, S.W. Bailey, ed., Applied Publishing, Wilmette, IL, 157-172.

Influence of Soluble Feed Proteins and Clay Additive Charge Density on Aflatoxin Binding in Ingested Feeds

467

Laird, D.A., Scott, A.D., & Fenton, T.E. 1989. Evaluation of the alkylammonium method of determining layer charge. Clays Clay Miner. 37, 41-46. Lipson, S.M., & Stotzky, G. 1984. Effect of proteins on Reovirus adsorption to clay minerals. Appl. Environ. Micro. 48, 525-530. MacEwan, D.M.C., & Wilson, M.J. 1984. Interlayer and intercalation complexes of clay minerals. pp. 197-248. In Brindley, G.W., & Brown, G. (eds.) Crystal Structures of Clay Minerals and their X-ray Identification. Mineralogical Society Monograph No. 5. Mineralogical Society, London. Magnoli, A.P., Tallone, L, Rosa, C.A.R., Dalcero, A.M., Chiacchiera, S.M., & Torres Sanchez, R.M. 2008. Commercial bentontites as detoxifier of broiler feed contaminated with aflatoxin. Appl. Clay Sci. 40, 63-71. Manahan, S.E. 2000. Environmental Chemistry. 7th edition. Lewis Publishers, New York. p. 237, 247. Mayland, H.F.,, Florence, A.R., Rosenau, R.C., Lazar, V.A., & Turner, H.A. 1975. Soil ingestion by cattle on semiarid range as reflected by titanium analysis of feces. J. Range Manage. 28, 448-452. Merck. 2001. The Merck Index, An Encyclopedia of Chemicals, Drugs and Biologicals. Merck Research Laboratories Division of Merck & Co., Inc. Whitehouse Station, NJ. p. 8290. Mermut, A.R., & Lagaly, G. 2001. Baseline studies of the Clay Minerals Society Source Clays: Layer-charge determination and characteristics of those minerals containing 2:1 layers. Clays Clay Miner. 49, 393-397. Miazzo, R., Rosa, C.A.R., De Queiroz Carvalho, E.C., Magnoli, C., Chiacchiera, S.M., Palacio, G., Saenz, M., Kikot, A., Basaldella, E., & Dalcero, A. 2000. Efficacy of synthetic zeolite to reduce the toxicity of aflatoxin in broiler chicks. Poult. Sci. 79, 1-6. Murphy, P.A., Hendrich, S., Landgren, C., & Bryant, C. M. (eds.). 2006. Food mycotoxins: An update. J. Food Sci. 71, R51-R65. Newman, A.C.D., & Brown, G. 1987. The chemical constitution of clays. In: Newman, A.C.D. (ed.) Chemistry of Clays and Clay Minerals. p. 56. Mineralogical Society Monograph No. 6, Longman Scientific & Technical, Essex, England. Odom, I.E. 1984. Smectite clay minerals: properties and uses. Phil. Trans. R. Soc. Lond. A 311, 391-409. Perez-Castells, R., Alvarez, A., Gavilanes, J., Lizarbe, M.A., Martinez Del Pozo, A., Olmo, N., & Santaren, J. 1985. Adsorption of collagen by sepiolite. In: Schultz, L.G., van Olphen, H., & Mumpton, F.A. (eds.) Proceedings of the International Clay Conference, Denver, CO 1985, pp. 359-362, The Clay Minerals Society, Bloomington, IN. Phillips, T.D., Kubena, L.F., Harvey, R.B., Taylor, D.R., & Heidelbaugh, N.D. 1988. Hydrated sodium aluminosilicate: a high affinity sorbent for aflatoxin. Poult. Sci. 67, 243-247. Phillips, T.D., Sarr, A.B., & Grant, P.G. 1995. Selective chemisorption and detoxification of aflatoxins by phyllosilicate clay. Natural Toxins 3, 204-213. Pimpukdee, K., Kubena, L.F., Bailey, C.A., Huebner, H.J., Afriyie-Gyawu, E., & Phillips, T.D. 2004. Aflatoxin-induced toxicity and depletion of hepatic vitamin A in young broiler chicks: Protection of chicks in the presence of low levels of Novasil Plus in the diet. Poult. Sci. 83, 737-744. Ralla, K., Sohling, U., Riechers, D., Kasper, C., Ruf, F., & Scheper, T. 2010. Adsorption and separation of proteins by a smectitic clay mineral. Bioprocess Biosyst. Eng. 33, 847861.

468

Aflatoxins – Biochemistry and Molecular Biology

Rao, S.B.N. & Chopra, R.C. 2001. Influence of sodium bentonite and activated charcoal on aflatoxin M1 excretion in milk of goats. Small Ruminant Research 41, 203-213. Rutenber, E., Katzin, B.J., Ernst, S., Collins, E.J., Mlsna, D., Ready, M.P., & Robertus, J.D. 1991. Crystallographic refinement of ricin to 2.5 Å. Proteins: Structure, Function, and Genetics, 10, 240-250. Scheideler, S.E. 1993. Effects of various types of aluminosilicates and aflatoxin B1 on aflatoxin toxicity, chick performance, and mineral status. Poult. Sci. 72, 282-288. Schell, T.C., Lindemann, M.D., Kornegay, E.T, Blodgett, D.J., & Doerr, J.A. 1993a. Effectiveness of different types of clay for reducing the detrimental effects of aflatoxin-contaminated diets on performance and serum profiles of weanling pigs. J. Anim. Sci. 71, 1226-1231. Schell, T.C., Lindemann, M.D., Kornegay, E.T., & Blodgett, D.J. 1993b. Effects of feeding aflatoxin-contaminated diets with and without clay to weanling and growing pigs on performance, liver function, and mineral metabolism. J. Anim. Sci. 71, 1209-1218. Sefa-Dedeh, S., Cornelius, B., Sakyi-Dawson, E., & Afoakwa, E. O. 2004. Effect of nixtamalization on the chemical and functional properties of maize. Food Chem. 86, 317-324. Seifert, L.E., Davis, J.P., Dorner, J.W., Jaynes, W.F., Zartman, R.E., & Sanders, T.H. 2010. Value-added processing of peanut meal: aflatoxin sequestration during protein extraction. J. Agric. Food Chem. 58, 5625-5632. Singer, A. 2002. Palygorskite and sepiolite. pp. 555-586. In Dixon, J.B., & Schulze, D.G. (eds.) Soil Mineralogy with Environmental Applications. SSSA Book Series, no. 7. Soil Science Society of America, Madison, WI. Squire, R.A. 1981. Ranking animal carcinogens: a proposed regulatory approach. Science 194, 877-880. Stryer, R. 1975. Introduction to protein structure and function. Chapter 2 In: Biochemistry. W.H. Freeman & Company. San Francisco, CA. Sweets, L.E., & Wrather, J.A. 2009. Aflatoxin in corn. University of Missouri, Delta Research Center. http://aes.missouri.edu/delta/croppest/aflacorn.stm. Thieu, N.Q., & Pettersson, H. 2008. In vitro evaluation of the capacity of zeolite and bentonite to adsorb aflatoxin B1 in simulated gastrointestinal fluids. Mycotoxin Research 24, 124-129. van Olphen, H., & Fripiat, J.J. 1979. Data Handbook for Clay Materials and other Nonmetallic Minerals. p. 198. Pergamon Press, New York. Vector Labs. 2011. Peanut agglutinin. www.vectorlabs.com/catalog.aspx? prodID=265. Vekiru, E., Fruhauf, S., Sahin, M., Ottner, F., Schatzmayr, G., & Krska, R. 2007. Investigation of various adsorbents for their ability to bind aflatoxin B1. Mycotoxin Research 23, 27-33. Veldman, A. 1992. Effect of sorbentia on carry-over of aflatoxin from cow feed to milk. Michwissenschaft 47, 777-780. Vincelli, P., Parker, G., & McNeill. 1995. Aflatoxins in corn. Cooperative Extension Service, University of Kentucky. www.ca.uky.edu/agc/pubs/id/id59/id59.pdf. Williams, J.H., Phillips, T.D., Jolly, P.E., Stiles, J.K., Jolly, C.M., & Aggarwal, D. 2004. Human aflatoxicosis in developing countries: a review of toxicology, exposure, potential health consequences, and interventions. Am. J. Clin. Nutr. 80, 1106-1122. Winfree, R.A., & Allred, A. 1992. Bentonite reduces measurable aflatoxin B1 in fish feed. The Progressive Fish-Culturist 54, 157-162. Zilinskas, R.A. Iraq’s biological weapons: The past as future? JAMA 278, 418-424.